Zymography 2017

Methods in
Molecular Biology 1626
Jeff Wilkesman
Liliana Kurz Editors
Zymography
Methods and Protocols
Methods in Molecular Biology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
Zymography
Methods and Protocols
Edited by
Jeff Wilkesman
Centre for Environmental, Biology and Chemistry Research, Facultad de Ciencias y Tecnología,
Universidad de Carabobo, Valencia, Venezuela
Liliana Kurz
Centre for Environmental, Biology and Chemistry Research, Facultad de Ciencias y Tecnología,
Universidad de Carabobo, Valencia, Venezuela
Editors
Jeff Wilkesman Liliana Kurz
Centre for Environmental, Biology Centre for Environmental, Biology
and Chemistry Research and Chemistry Research
Facultad de Ciencias y Tecnología Facultad de Ciencias y Tecnología
University of Carabobo University of Carabobo
Valencia, Venezuela Valencia, Venezuela
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-7109-1 ISBN 978-1-4939-7111-4 (eBook)
DOI 10.1007/978-1-4939-7111-4
Library of Congress Control Number: 2017943128
© Springer Science+Business Media LLC 2017

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Dedication
To our children, Jarod and Kylie

To our other children: our students
In Memoriam
To Prof. Ricardo Maldonado, former Rector-President of the University of Carabobo, who

untimely passed away the night before Christmas.
vii
Preface
Zymography, the technique where enzyme activity is detected on electrophoretic gels, has
received increasing attention in recent years. This book updates the information concerning
the latest techniques and protocols associated with zymography. The methods described are
intended to reach a broad variety of researchers from the fields of biological and medicinal
science. Isozymes and allozymes, used as gene markers, enable advances in enzymology,
molecular evolution, and genetics. Many fields will benefit from a bibliographical collection
regarding zymography, including clinical and diagnostic medicine, medical genetics, agricul-
tural entomology, genetic monitoring of environmental pollution, and forensic science.
The main purpose of this volume is to bring together some of the most recent and
broad zymography methods currently used. The book is of value not only to experts in the
field but also to the incoming new scientists willing to learn and perform electrophoretic
zymography.
Chapters have been written following the classic format employed in the Methods and
Molecular Biology series. All chapters initiate with a brief description of the basic theory
behind the method being analyzed. The Materials section lists all chemicals, reagents,
buffers, and other materials necessary for the correct performance of the experiments.
The Methods section includes a detailed stepwise description of the protocol. Deatails
regarding problem shooting, tips, hints, and advices for key steps in experimentation are
given in the Notes section, thus complementing the Method section.
Valencia, Venezuela Jeff Wilkesman

Liliana Kurz
ix
Acknowledgments
We would like to thank all the chapter authors and coauthors for their motivation and trust
in submitting their chapters. We are extremely grateful for the opportunity given by John
M. Walker (University of Hertfordshire, UK) and all his support and advice throughout the
different stages in the elaboration of this volume.
It is an honor for us to make this contribution to the world, and yet, this would not
have been possible without the contribution of our first mentor, José Bubis (University
Simón Bolívar, Venezuela), who taught us the basics and introduced us to this wonderful
technique during our undergraduate performance.
I am especially grateful to Philipp Wiedemann and Lasse Greiner, from the University
of Applied Sciences Mannheim (Germany), for facilitating time and space for the writing
and editing of this book.
Preparation and editing of the manuscript was partially supported by Universidad de
Carabobo, Venezuela, during the sabbatical year of the authors.
This work was also supported by the National Research, Development and Innovation
Office (NKFIH, TET_15_IN-1-2016-0068).
Jeff Wilkesman
Liliana Kurz
xi
Contents
Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
In Memoriam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Part I Introduction
1 Zymography Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Jeff Wilkesman and Liliana Kurz
Part II Endopeptidase Zymography

2 Serine Protease Zymography: Low-Cost, Rapid, and Highly Sensitive
RAMA Casein Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Hidetaro Yasumitsu
3 Cysteine Protease Zymography: Brief Review . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Jeff Wilkesman
4 Aspartic Protease Zymography Case Study: Detection of Fungal Acid
Proteases by Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Gavin Kernaghan and Michael Mayerhofer
5 Detection of Aspartic Proteinase Activities Using Gel Zymography . . . . . . . . . . 43
Handunge Kumudu Irani Perera
6 MMP Activity Detection in Zymograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Péter Bencsik, Monika Bartekova, Anikó Görbe, Krisztina Kiss,
János Pálóczi, Jana Radosinska, Gergő Szűcs, and Péter Ferdinandy
7 Characterization of Novel Collagenolytic Proteases . . . . . . . . . . . . . . . . . . . . . 71
Goran Mucić, Brankica Rašković, and Natalija Polović
8 Zymography as a Research Tool in the Study of Matrix Metalloproteinase
Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Zongli Ren, Juanjuan Chen, and Raouf A. Khalil
9 Detection and Characterization of Bacterial Proteinases Using Zymography . . . 103
Madathiparambil G. Madanan and Ambili Mechoor
10 A Sensitive, Rapid, and Specific Technique for the Detection
of Collagenase Using Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Shivcharan Prasad and Ipsita Roy
Part III Reverse Zymography and In Situ Zymography

11 Reverse Zymography: Overview and Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Kanika Sharma and Debasish Bhattacharyya
xiii
xiv Contents
12 Cell In Situ Zymography: Imaging Enzyme–Substrate Interactions . . . . . . . . . 133

Aastha Chhabra and Vibha Rani
Part IV 2D Zymography

13 Examination of Gelatinase Isoforms in Rodent Models
of Acute Neurodegenerative Diseases Using Two-Dimensional Zymography . . 147
Shanyan Chen, Fanjun Meng, Zhenzhou Chen, Zhe Qu, Jiankun Cui,
and Zezong Gu
14 Two-Dimensional Zymography of Proteases from Steatotic Duck Liver . . . . . . 157
Jeff Wilkesman, María Fernanda Padrón, Liliana Kurz,
and Hervé Rémignon
Part V Special Zymography Cases

15 Simultaneous Detection of Activity and Relative Molecular Mass
of Adenylate Kinases After SDS-PAGE and Blotting . . . . . . . . . . . . . . . . . . . . . 169
Silvia Ravera and Isabella Panfoli
16 Silver-Stained Fibrin Zymography: Separation of Proteases
and Activity Detection Using a Single Substrate-Containing Gel . . . . . . . . . . . 179
Chang-Su Park, Dae-Ook Kang, and Nack-Shick Choi
17 Zymography Methods to Simultaneously Analyze Superoxide Dismutase
and Catalase Activities: Novel Application for Yeast Species Identification . . . . . 189
Esther Gamero-Sandemetrio, Rocío Gómez-Pastor, and Emilia Matallana
18 Detection of Guaiacol Peroxidase on Electrophoretic Gels . . . . . . . . . . . . . . . . 199
Diana Castro, Lellys M. Contreras, Liliana Kurz, and Jeff Wilkesman
19 In Situ Demonstration and Characteristic Analysis of the Protease
Using Substrate Immersing Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
HaiLun He, Hao Li, and Dan Liu
20 Use of Zymography in Trypanosomiasis Studies . . . . . . . . . . . . . . . . . . . . . . . . 213
Jéssyka Fernanda Santiago Monte, Cláudia Jassica Gonçalves Moreno,
Joana Patrícia Molato Figueiredo Lopes Monteiro,
Hugo Alexandre de Oliveira Rocha, Aline Rimoldi Ribeiro,
and Marcelo Sousa Silva
21 Zymography in Multiwells for Quality Assessment of Proteinases . . . . . . . . . . . 221
Ambili Mechoor and Madathiparambil G. Madanan
22 Visualization of Enzyme Activities in Earthworm Biopores
by In Situ Soil Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Bahar S. Razavi, Duyen Hoang, and Yakov Kuzyakov
23 Multiplex Cathepsin Zymography to Detect Amounts
of Active Cathepsins K, L, S, and V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Manu O. Platt
24 Transfer Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Daniel Pan, Karl A. Wilson, and Anna Tan-Wilson
25 Sequential Detection of Thermophilic Lipase and Protease by Zymography . . . 271
Liliana Kurz, Zully Hernández, Lellys M. Contreras, and Jeff Wilkesman
Contents xv
26 Calpain Zymography: General Methodology and Protocol . . . . . . . . . . . . . . . . 279

Kevin K.W. Wang
27 CTAB Zymography for the Analysis of Aspartic Proteases
from Marine Sponges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Oscar González and Jeff Wilkesman
28 Zymography Detection of a Bacterial Extracellular Thermoalkaline
Esterase/Lipase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
María Tapizquent, Marleny Fernández, Georgina Barreto,
Zully Hernández, Lellys M. Contreras, Liliana Kurz, and Jeff Wilkesman
29 Amylase Zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Adarelys Andrades and Lellys M. Contreras
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Contributors
Adarelys Andrades • University of Leipzig, Leipzig, Germany; Centre for Environmental,

Biology and Chemistry Research, Facultad de Ciencias y Tecnología, Universidad de
Carabobo, Valencia, Venezuela
Georgina Barreto • Centre for Environmental, Biology and Chemistry Research, Facultad
de Ciencias y Tecnología, Universidad de Carabobo, Valencia, Venezuela
Monika Bartekova • Institute for Heart Research, Slovak Academy of Sciences, Bratislava,
Slovak Republic; Institute of Physiology, Faculty of Medicine, Comenius University,
Bratislava, Slovak Republic
Péter Bencsik • Department of Biochemistry, Faculty of Medicine, University of Szeged,
Szeged, Hungary; Pharmahungary Group, Szeged, Hungary
Debasish Bhattacharyya • Division of Structural Biology and Bioinformatics,
CSIR-Indian Institute of Chemical Biology, Kolkata, India
Diana Castro • Centre for Environmental, Biology and Chemistry Research, Facultad de
Ciencias y Tecnología, Universidad de Carabobo, Valencia, Venezuela
Juanjuan Chen • Vascular Surgery Research Laboratory, Division of Vascular and
Endovascular Surgery, Brigham and Women’s Hospital and Harvard Medical School,
Boston, MA, USA
Shanyan Chen • Department of Pathology and Anatomical Sciences, University of Missouri
School of Medicine, Columbia, MO, USA; Interdisciplinary Neuroscience Program,
University of Missouri, Columbia, MO, USA
Zhenzhou Chen • Department of Pathology and Anatomical Sciences, University of
Missouri School of Medicine, Columbia, MO, USA
Aastha Chhabra • Peptide and Proteomics Division, Defence Institute of Physiology and
Allied Sciences (DIPAS), DRDO, Timarpur, Delhi, India; Department of Biotechnology,
Jaypee Institute of Information Technology, Noida, Uttar Pradesh, India
Nack-Shick Choi • Department of Biochemistry & Health Science, Changwon National
University, Changwon, Changwon-si, Republic of Korea; Careside Co., Ltd.,
Seongnam-si, Gyunggi-do, Republic of Korea
Lellys M. Contreras • Centre for Environmental, Biology and Chemistry Research,
Facultad de Ciencias y Tecnología, Universidad de Carabobo, Valencia, Venezuela
Jiankun Cui • Department of Pathology and Anatomical Sciences, University of Missouri
School of Medicine, Columbia, MO, USA; Harry S. Truman Memorial Veterans’ Hospital,
Columbia, MO, USA
Péter Ferdinandy • Pharmahungary Group, Szeged, Hungary; Department of
Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary
Marleny Fernández • Centre for Environmental, Biology and Chemistry Research,
Esther Gamero-Sandemetrio • Departamento de Biotecnología, Instituto de Agroquímica
y Tecnología de Alimentos, CSIC, Valencia, Spain
Rocío Gómez-Pastor • Departamento de Biotecnología, Instituto de Agroquímica y
Tecnología de Alimentos, CSIC, Valencia, Spain; Departament de Bioquímica i Biologia
Molecular, Universitat de València, València, Spain
xvii
xviii Contributors
Oscar González • Centre for Environmental, Biology and Chemistry Research, Facultad

Anikó Görbe • Department of Biochemistry, Faculty of Medicine, University of Szeged,
Szeged, Hungary; Pharmahungary Group, Szeged, Hungary
Zezong Gu • Department of Pathology and Anatomical Sciences, University of Missouri
School of Medicine, Columbia, MO, USA; Harry S. Truman Memorial Veterans’
Hospital, Columbia, MO, USA
HaiLun He • School of Life Sciences, State Key Laboratory of Medical Genetics, Central
South University, Changsha, China
Zully Hernández • Centre for Environmental, Biology and Chemistry Research,
Duyen Hoang • Department of Soil Science, Vietnam Nation University of Forestry,
Hanoi, Vietnam
Dae-Ook Kang • Department of Biochemistry & Health Science, Changwon National
University, Changwon, Republic of Korea
Gavin Kernaghan • Department of Biology, Mount Saint Vincent University, Halifax, NS,
Canada
Raouf A. Khalil • Vascular Surgery Research Laboratory, Division of Vascular and
Boston, MA, USA
Krisztina Kiss • Department of Biochemistry, Faculty of Medicine, University of Szeged,
Szeged, Hungary
Liliana Kurz • Centre for Environmental, Biology and Chemistry Research, Facultad de
Ciencias y Tecnología, University of Carabobo, Valencia, Venezuela
Yakov Kuzyakov • Department of Agricultural Soil Science, University of Göttingen,
Göttingen, Germany; Department of Soil Science of Temperate Ecosystems, University of
Göttingen, Göttingen, Germany
Hao Li • College of Life Science and Technology, Beijing University of Chemical Technology,
Beijing, China
Dan Liu • School of Life Sciences, State Key Laboratory of Medical Genetics, Central South
University, Changsha, China
Madathiparambil G. Madanan • Regional Medical Research Centre, Indian Council of
Medical Research, Port BlairAndaman & Nicobar Islands, India
Emilia Matallana • Departamento de Biotecnología, Instituto de Agroquímica y
Tecnología de Alimentos, CSIC, Valencia, Spain; Departamento de Biotecnología,
Instituto de Agroquímica yTecnología de Alimentos, CSIC, Valencia, Spain;
Departament de Bioquímica i Biologia Molecular, Universitat de València, Valencia,
Spain
Michael Mayerhofer • Department of Biology, Mount Saint Vincent University, Halifax,
NS, Canada
Ambili Mechoor • Department of Biotechnology Engineering, Sahrdaya College of
Engineering and Technology, Thrissur, Kerala, India
Fanjun Meng • Department of Pathology and Anatomical Sciences, University of Missouri
School of Medicine, Columbia, MO, USA
Jéssyka Fernanda Santiago Monte • Programa de Pós-graduação em Bioquímica, Centro
de Biociências, Universidade Federal do Rio Grande do Norte, Natal, Brazil
Joana Patrícia Molato Figueiredo Lopes Monteiro • Global Health and Tropical
Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa,
Lisbon, Portugal
Contributors xix
Cláudia Jassica Gonçalves Moreno • Programa de Pós-graduação em Bioquímica,

Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, Brazil
Goran Mucić • Department of Biochemistry, Faculty of Chemistry, University of Belgrade,
Belgrade, Serbia
María Fernanda Padrón • Centre for Environmental, Biology and Chemistry Research,
János Pálóczi • Pharmahungary Group, Szeged, Hungary
Daniel Pan • Department of Biological Sciences, State University of New York,
Binghamton, NY, USA; Department of Systems Pharmacology and Translational
Therapeutics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA,
USA
Isabella Panfoli • Biochemistry Laboratory, Pharmacy Department, University of Genova,
Genova, Italy
Chang-Su Park • Department of Food Science & Technology, Catholic University of Daegu,
Daegu, Republic of Korea
Handunge Kumudu Irani Perera • Department of Biochemistry, Faculty of Medicine,
University of Peradeniya, Peradeniya, Sri Lanka
Manu O. Platt • Wallace H. Coulter Department of Biomedical Engineering, Georgia
Institute of Technology and Emory University, Atlanta, GA, USA
Natalija Polović • Department of Biochemistry, Faculty of Chemistry, University of
Belgrade, Belgrade, Serbia
Shivcharan Prasad • Department of Biotechnology, National Institute of Pharmaceutical
Education and Research, Mohali, Punjab, India
Zhe Qu • Department of Pathology and Anatomical Sciences, University of Missouri School
of Medicine, Columbia, MO, USA
Jana Radosinska • Institute for Heart Research Slovak Academy of Sciences, Bratislava,
Slovak Republic; Institute of Physiology, Faculty of Medicine, Comenius University,
Bratislava, Slovak Republic
Vibha Rani • Department of Biotechnology, Jaypee Institute of Information Technology,
Noida, Uttar Pradesh, India
Brankica Rašković • Department of Biochemistry, Faculty of Chemistry, University of
Belgrade, Belgrade, Serbia
Silvia Ravera • Biochemistry Laboratory, Pharmacy Department, University of Genova,
Genova, Italy
Bahar S. Razavi • Department of Agricultural Soil Science, University of Göttingen,
Göttingen, Germany
Hervé Rémignon • GenPhySE, Université de Toulouse, INRA, INPT, ENVT, Castanet
Tolosan, France
Zongli Ren • Vascular Surgery Research Laboratory, Division of Vascular and
Boston, MA, USA
Aline Rimoldi Ribeiro • Departmento de Parasitologia, Universidade Estadual de
Campinas, Campinas, Brazil
Hugo Alexandre de Oliveira Rocha • Programa de Pós-graduação em Bioquímica,
Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, Brazil
Ipsita Roy • Department of Biotechnology, National Institute of Pharmaceutical Education
and Research, S.A.S. Nagar, Mohali, Punjab, India
xx Contributors
Kanika Sharma • Division of Structural Biology and Bioinformatics, CSIR-Indian

Institute of Chemical Biology, Kolkata, India
Marcelo Sousa Silva • Programa de Pós-graduação em Bioquimica, Centro de
BiocienciasUniversidade Federal do Rio Grande do Norte, Natal, Brazil; Global Health
and Tropical Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de
Lisboa, Lisbon, Portugal; Departamento de Analises Clinicas e Toxicologicas, Centro de
Ciencias da Saude, Universidade Federal do Rio Grande do Norte, Natal, Brazil
Gergő Szűcs • Department of Biochemistry, Faculty of Medicine, University of Szeged,
Szeged, Hungary
Anna Tan-Wilson • Department of Biological Sciences, State University of New York,
Binghamton, NY, USA
María Tapizquent • Centre for Environmental, Biology and Chemistry Research, Facultad
Kevin K.W. Wang • Program for Neurotrauma, Neuroproteomics & Biomarker Research,
Department of Psychiatry, University of Florida, Gainesville, FL, USA; Department of
Neuroscience, University of Florida, Gainesville, FL, USA; Department of Chemistry,
University of Florida, Gainesville, FL, USA
Jeff Wilkesman • Centre for Environmental, Biology and Chemistry Research, Facultad de
Ciencias y Tecnología, University of Carabobo, Valencia, Venezuela
Karl A. Wilson • Department of Biological Sciences, State University of New York,
Binghamton, NY, USA
Hidetaro Yasumitsu • Expert Laboratory for Life Environments (ELLE), Yokohama City
University, Yokohama, Japan; Division of Pharmacy, Kamimach Hospital, Kochi, Japan
Part I
Introduction
Chapter 1
Zymography Principles
Jeff Wilkesman and Liliana Kurz
Abstract
Zymography, the detection, identification, and even quantification of enzyme activity fractionated by gel
electrophoresis, has received increasing attention in the last years, as revealed by the number of articles
published. A number of enzymes are routinely detected by zymography, especially with clinical interest. This
introductory chapter reviews the major principles behind zymography. New advances of this method are basically
focused towards two-dimensional zymography and transfer zymography as will be explained in the rest of the
chapters. Some general considerations when performing the experiments are outlined as well as the major
troubleshooting and safety issues necessary for correct development of the electrophoresis.
Key words Electrophoresis, Enzymes, Zymography, Troubleshooting
1 Introduction
Zymography is the technique based on sodium dodecyl sulfate poly-

acrylamide gel electrophoresis (SDS-PAGE) which enables to detect
enzymatic activity. The substrate may be copolymerized within the
polyacrylamide gel matrix, but could also be submerged in a sub-
strate solution or can be overlaid with another gel containing the
substrate [1]. Samples submitted to zymography are generally pre-
pared by the standard SDS-PAGE treatment buffer. However, non-
reducing conditions must prevail at all times, viz., samples are not
heated and there is no reducing agent [2-mercaptoethanol, dithio-
threitol (DTT)] present. After the electrophoresis has been run,
SDS is soaked out from the gel (zymogram) by incubation in a non-
buffered Triton X-100 (or other detergent, e.g., Tween-20, etc.).
After that, an appropriate activation buffer is selected and the zymo-
gram is incubated for a determined length of time and temperature,
depending on the type of enzyme being assayed and the type of
substrate being degraded. Finally, the zymogram is stained. Staining
procedures may be direct, obtaining colored bands with a translu-
cent background; or may be indirect, and areas of digestion are dis-
tinguished as pale bands under a dark background.
Jeff Wilkesman and Liliana Kurz (eds.), Zymography: Methods and Protocols, Methods in Molecular Biology, vol. 1626,
DOI 10.1007/978-1-4939-7111-4_1, © Springer Science+Business Media LLC 2017
3
4 Jeff Wilkesman and Liliana Kurz
2 Classification
Different types of zymography exist, according to the type of

enzyme. There are over 1000 different methods published for the
detection of more than 400 different enzymes [2].
However three main types of zymography may be postu-
lated [3, 4]:
–– In gel zymography (IGZ): briefly, the SDS-PAGE is employed
to separate the enzyme-containing samples and is later over-
laid on an indicator gel. A variant of this type of zymography
is the direct incorporation of the substrate to the gel matrix
[4].
–– In situ zymography (ISZ): based on zymography using SDS-
PAGE, the sample—a tissue section or a cell preparation—is
brought in contact with the substrate employing a photo-
graphic emulsion-containing gelatin (or a fluorescence-
labeled protein substrate). After exposure, enzyme activity
appears as white spots in a dark background (or as black
spots in a fluorescent background). Therefore, exact local-
ization of proteinase activity is achieved. As asset, endoge-
nous enzymes in their biological contexts are visualized (see
part III of this book).
–– In vivo zymography (IVZ): relying on the use of protease-
activated fluorogenic probes bearing different fluorophores,
this type of zymography allows detection of diverse enzymes
simultaneously. It has been used for the mapping of MMP
activity patterns in intact organisms, e.g., zebrafish, as well as
in roots from maize and lupine [5–7].
Based upon the type of detection performed, it is generally
encountered as either a chromogenic or a fluorogenic reaction respon-
sible for enzyme visualization on the gel. Some of the most common
chromogenic reactions rely upon the following chemistry [2]:
–– Reduction of tetrazolium salts (viz. NBT).
–– Coupling diazonium salts (e.g., Fast Blue, Fast Red, etc.).
–– Change in local pH (use of pH indicators for detection).
–– Formation of orthophosphate or pyrophosphate.
–– Formation of hydrogen peroxide (detection performed with
redox dyes).
–– Production of carbon dioxide.
–– Use of chromogenic substrates (i.e., derivatives of para-
nitrophenol or para-nitroaniline).
Besides chromogenic detection, fluorogenic detection is pos-
sible. The natural fluorochromes NADH and NADHP are widely
Zymography Principles 5
used, as after oxidation they lose their fluorescence. Derivatives of

4-methylumbelliferone are also widely used, especially for hydro-
lase detection [2].
Some variations of IGZ are described in the upcoming chap-
ters. Worth mentioning here are electrophoretic transfer zymogra-
phy [8]. Here, the enzyme is resolved in a polyacrylamide gel
without protein substrate, and afterward the enzyme in the gel is
transferred to a receptor gel that does contain the substrate. This
step circumvents the issue of some enzymes exhibiting a lower
mobility value when migrating in a substrate gel. In transfer
zymography, the mobilities of the enzyme are comparable to those
obtained by non-reducing SDS-PAGE.
Another variant is two-dimensional zymography (2DZ). In this
case, the sample to be studied is first submitted to isoelectric focus-
ing on IPG-strips. Later, the strip is submitted to conventional
IGZ, gaining an extra dimension in the analysis, as now proteins
are separated not only by molecular mass, but also according to
their isoelectric point [9]. Not less important is another variation
of IGZ called reverse zymography, which enables the detection of
enzyme inhibitors. For this purpose, substrate and enzyme are
both copolymerized within the gel matrix. After proper reactiva-
tion of the enzyme, the copolymerized substrate is evenly degraded
except in those spots where an inhibitor is present preventing
enzyme action. After staining, inhibitor location can be visualized
as bands containing intact substrate [10].
3 Advantages of PAGZ
Zymography allows a number of advantages when analyzing unpu-

rified enzyme preparations, namely [2, 4]:
–– All kinds of biological samples may be analyzed, from cell
and tissue extracts, to culture fluids, whole blood, plasma,
serum as well as other complex body or lavage fluids, tissue
sections, and even whole organisms [2, 3].
–– Detection and identification of isozymes in crude cell
extracts.
–– Functional properties of isozymes directly in gel.
–– Quantitative and qualitative information is given by means
of estimating the molecular mass and the isoelectric point of
the enzyme.
–– Monitoring specific and non-specific enzymatic activities in
complex biological and clinical samples.
–– Discrimination of enzymes with similar and/or overlapping
substrate specificities.
–– Possible determination of subunit structure.
As can be seen, zymography provides general and specific and

useful information in the broad area of pure and applied biochem-
istry. Clinical and diagnostic medicine has been more impacted by
zymography. Topics like cancer, heart and vascular diseases, and
immunological inflammatory diseases have standard zymography
assays in their procedures. Zymography is also present in animal
science, like the study of proteases in chicken, duck, and others
[11] and agricultural and plant science [6, 7, 12], forensic science,
etc. [2, 4].
In comparison with other biotechnological techniques, zymog-
raphy remains a very simple and powerful procedure for the parti-
tion, identification, and characterization of gene products.
Proteomic has a huge ally in zymography, given its advantages.
2DZ, as a result of 2DE and transfer zymography, gives valuable
material to be further analyzed by mass spectrometry. The combi-
nation of flexible enzyme assay techniques with electrophoresis
represents a powerful tool to cope with the rising demand of high-
throughput screening coming from functional genomics, protein
engineering, and combinatorial chemistry. Given the impact
exerted by zymography, it is not difficult to see the importance to
further continue the research in new and enhanced methodologies
for enzymatic analysis.
4 Final Remarks
The choice of a given zymographic method may depend on highly

diverse variables. Compatibility of reagents with the matrix and the
enzyme, sensitivity, protein concentration, time-consuming proto-
cols, quantification of results, band stability throughout time,
costs, all these are elements to be considered before adopting a
protocol. Beyond this, it may happen that even after thoughtful
consideration, zymography fails in giving appropriate results. Here,
we present some general conditions when the general procedure
by some reason does not work. Given the amount of steps involved
in the general zymography procedure, it is advised to analyze each
step separately.
4.1 Troubleshooting 1. Support media: although the enzyme detection is not neces-
sarily dependent on the support medium used, its choice is
important as it will define the quality of the zymography. The
most popular is polyacrylamide; still other media are also pop-
ular depending on the tested enzyme, viz., starch, cellulose
acetate [13].
2. Gel staining: for some given enzymes, more than one method
may be available. Choosing the most appropriate one may be
difficult and can depend on variables such as length of the
procedure, chemicals involved, mode of application of the

staining solution, and election of appropriate controls to test for
positive and negative results. Staining solution must always be
abundant. The amount of solution varies according to the size of
the gel and the size of the tray where staining is taking place. This
is particularly important, as some staining procedures are costly.
Instead of submerging the gel in a tray containing the staining
solution, an agar or a filter paper soaked with the solution can be
overlaid. Direct application of drops of the solution onto the
zymogram with a pippet is also an option sometimes [2].
3. Staining conditions: revise if a specific temperature is needed
for reaction (generally 37 °C), and if luminic conditions are to
be avoided. Keep track of the incubation time. Generally, stain-
ing reactions proceed in the first 10 min. However, there are
cases where a 24–48 h incubation time is needed. This in turn
implies how specific the enzyme is towards the substrate. For
proteases, Coomassie blue is by far the most employed staining
method; however, other stains may be used (protein silver
stain, Congo red staining for cellulose, and Amido black stain).
With the increasing versatility of gel scanners (including not
only visible and UV lamps, but now also fluorescent detec-
tion), the range of zymography applications is boundless. New
staining protocols are constantly being published (see Chapter
2), like the new RAMA stain [14].
4. Solution preparation: when working, effort must be made to
prepare all solutions fresh. Indeed, it is possible to make stock
solutions for gel polymerization and preserve them in cold.
However, these solutions after 14 days tend to get contami-
nated by microorganisms, especially buffers. Many staining
solutions must be prepared fresh and solution recycling is not
advised in many cases. Of course, each experimenter must in
some way find out if a determined solution may or may not be
reused, and if, under which circumstances can it be preserved
until its next use.
5. Running conditions: pre-running gels may be useful to elimi-
nate possible non-polymerized elements from the gel that could
potentially interfere in enzyme activation. Generally, it is advised
to run the gel at 4 °C, especially if the enzyme is thermolabile.
During electrophoresis, it has been suggested to run the gel at
20 mA per gel. If running condition is set at constant current,
the voltage will increase with time reaching values eventually
over 200 V. On the other hand, if the power supply is set to run
at constant voltage (usually 150 V), the initial current will typi-
cally be around 35 mA and will drop with the pass of time,
reaching 10 mA by the end of the run. Either way, it is suggested
to select running conditions which will allow less stress to the
enzyme, i.e., lower current and more running time.
6. pH conditions: this variable is hardly enough studied. Given

the variation of pH with temperature, buffer concentration
and pH range must be carefully controlled. The final pH of the
staining solution must fulfill the criteria demanded by the pH
optimum of the enzyme, the pH of the reaction taking place in
the staining procedure, the pH of the buffer employed in gel
preparation, and if there is a coupled reaction in the staining,
then the pH optimum of the coupled reaction.
7. Buffers: besides checking the pH of the buffer at the real tem-
perature where it is going to be used, its buffering capacities
may also be affected if the concentration is too low. Generally,
50 mM buffer should be the least concentration used to assure
proper buffering conditions.
8. Substrate: as there is a huge diversity of substrate, either natu-
ral or synthetic, it is wise to check its stability. Solubility is
sometimes a problem. If incorporated to the matrix, it must be
assured that the substrate will not migrate when electric field is
applied. Substrate must be provided in a high purity state and
its optimal concentration must be previously determined.
Typical substrates for protease analysis are gelatin, casein, col-
lagen, and fibrin [15].
9. Detergent removal: it is important to decide which detergent will
be utilized in the washing buffer in IGZ [3]. Structure of the
detergent’s hydrophilic head group and its critical micelle con-
centration (CMC) affect the removal of SDS. Typically, 1 h incu-
bation with a 1–2.5% (v/v) Triton X-100% is used. However,
Tween-20 or Tween-80 may also be used satisfactorily.
10. Reactivation stage: after SDS removal, the enzyme is ready to
be refolded and gain biological activity again. Besides checking
pH and temperature conditions, the reactivation buffer selected
should be also able to fulfill the requirement of any necessary
cofactor. Depending on the enzyme, typical cofactors are diva-
lent cations (Ca2+, Mg2+), NAD+, etc.
11. Enzyme biological source: typical mistakes can be traced back
to homogenate preparation. Related questions to be formu-
lated are: how fresh is your sample? Where was it stored and for
how long? Was it prepared in the appropriate buffers? Is the
concentration high enough? Was a parallel enzyme test in test
tube performed before electrophoresis?
12. Some frequently asked questions regarding failure in the pro-
cedure are:
–– Are all reagents in the staining solution present? Check the
protocol and revise preparation of each solution. Many times,
miscalculation occurs either in weighing or in dissolving.
–– Are the reagents stable in time? Check the stability of all
reagents. Some of them are light-sensible and are spontaneously
hydrolyzed under inappropriate pH conditions. An inadequate

storage can occur (left out the fridge, exposed to light, or
contaminated).
–– Was there a pH alteration? Measure pH of the staining
solution and contrast with protocol requirements. Check pH
of all solutions involved. Prepare fresh solutions if needed.
–– Could there be enzyme inhibitors present in the solutions?
Indeed, this is possible, especially if experiments are done in
the field. Use always distilled water for experiments. Other
enzyme inhibitors may be present in the gel matrix.
Sometimes, reactivation of the enzyme after electrophoresis
does not happen.
4.2 Safety Many of the chemical reagents used during the zymography proce-
Regulations dure are toxic, carcinogenic, and skin irritants. Therefore, it is wise
to follow a strict security code assuming that all substances are
potentially hazardous. The use of gloves throughout the entire
experiment is mandatory. Dust mask shall be worn when handling
chemicals, especially solids. Solution preparation may produce
vapors; use the safety hood at all times. Do not discard the used
solutions in the sink. Check first for the environmental risks of each
of the chemicals and then proceed to discard appropriately. The
ten principles of green chemistry shall be followed at all times. As
many solutions will be prepared, label all the containers, indicating
expiry date of the solutions.
Remember to keep all areas clean and tidy. Clean up immedi-
ately any spills on the bench. After completing experiments, dis-
card gloves and wash hands thoroughly. Remember that the lab is
not a kitchen! Do not keep food in the lab fridge.
Acknowledgments
We would like to thank Prof. Dr. J. Bubis and M. Calabokis

(University Simón Bolívar) for their unconditional support during
all this time. We thank Prof. Dr. W.E.G. Müller and H.C. Schröder,
for opening the doors of their labs to host our research stay. We
appreciate the support of the University of Carabobo.
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Part II
Endopeptidase Zymography
Chapter 2
Serine Protease Zymography: Low-Cost, Rapid, and Highly

Sensitive RAMA Casein Zymography
Hidetaro Yasumitsu
Abstract
To detect serine protease activity by zymography, casein and CBB stain have been used as a substrate and
a detection procedure, respectively. Casein zymography has been using substrate concentration at 1 mg/
mL and employing conventional CBB stain. Although ordinary casein zymography provides reproducible
results, it has several disadvantages including time-consuming and relative low sensitivity. Improved casein
zymography, RAMA casein zymography, is rapid and highly sensitive. RAMA casein zymography com-
pletes the detection process within 1 h after incubation and increases the sensitivity at least by tenfold. In
addition to serine protease, the method also detects metalloprotease 7 (MMP7, Matrilysin) with high
sensitivity.
Key words Casein zymography, CBB stain, RAMA stain, Electrophoresis, Serine protease, Proteinase,
SDS-PAGE, MMP7
1 Introduction
Serine proteases are known to be one of the major enzymes spread-

ing over all living things and to play important roles not only in
their life but also in human health. Viral serine proteases are
required for their propagation and aimed to be an important target
for therapy [1]. Bacterial serine proteases are reported to be
involved in their growth and virulence [2, 3]. Fungal serine prote-
ases are known to be required for their life and induce allergy and
inflammation in human body [4]. Plant serine proteases also play
important roles in their life [5]. In relation to human life, snake
venom is one of the most important proteases sources and has
been studied and revealed to disturb coagulation and fibrinolysis in
the prey systems [6, 7].
Human serine proteases have been studied for many years and
revealed to play many important roles in normal and pathogenic
processes including digestive system [8], defense [9], inflamma-
tion [10], allergy [11], cardiac function [12], and behavior [13].
13
14 Hidetaro Yasumitsu
To detect serine protease activities, protein substrate zymogra-

phy is one of the major activity assays and frequently used. Casein and
gelatin have been used as the protein substrates. Recently, several
variations of zymography and new platforms for activity assay were
reported [14–16]. However, since casein and gelatin zymography are
able to detect many endopeptidase species at low cost and with decent
sensitivity, they have been still used in many laboratories.
In casein zymography, samples are usually separated on a gel
containing 1 mg/mL casein, followed then by a conventional CBB
staining step [17, 18]. Although these conditions and procedures
provide reproducible results, the zymography is time-consuming
(usually overnight destaining) and its sensitivity is relatively low.
In that method, one of the major reasons for low sensitivity
might be attributed to high concentration of substrate in a separa-
tion gel. Since proteases with weak activity might digest only small
amount of substrate during the incubation period, i.e., within a
protease-existing area, undigested substrates still might remain and
then the residual substrate might be stained as substantially blue,
and therefore, the area having weak protease activity might seem
unclear and might be difficult to recognize as a band. When a lower
concentration of substrate is employed, most of the substrate in a
weak protease-existing area might be digested thoroughly.
However, by using the low substrate concentration, the contrast
between digested and undigested area might decrease, and there-
fore, all the active bands might become obvious.
To overcome the above-mentioned problems, low concentra-
tion substrate and high sensitive protein-staining methods (CGP
stain and RAMA stain), which have recently been established by us
[19, 20], are combined. Resulted new method of RAMA casein
zymography, which employed RAMA stain in combination with
low casein concentration, improves the detection sensitivities of
trypsin and MMP7 by at least tenfold [21]. In addition, stain and
destain processes complete within 1 h after incubation.
2 Materials
All the reagents used are reagent grade or analytical grade. The water
used for preparation of reagents and for gel washing is Milli Q water
(MQW: 18.2 mho). Importantly, all of the prepared aqueous solu-
tions should be paid attention to for the contamination by microor-
ganisms including bacteria and fungi. Once such microorganisms
grow in a solution, it is contaminated with lot of proteases and arti-
ficial protease bands appear on the zymogram. RAMA casein zymog-
raphy is sensitive enough to detect them. When slight amounts of
precipitates appear or are noticed existing in your aqueous solution,
discard it and prepare another fresh one.
RAMA Casein Zymography 15
2.1 Electrophoresis 1. Thirty percent of acrylamide stock solution for separation gel
(a mixture of 29.2% acrylamide and 0.8% bisacrylamide) is pre-
pared by dissolving them and stored at 4 °C in a bottle covered
with aluminum foil for avoiding light (see Note 1).
2. Ten percent of acrylamide stock solution for stacking gel (a mix-
ture of 10% acrylamide and 2.5% bisacrylamide) is prepared by
dissolving them and stored at 4 °C, avoiding light (see Note 2).
3. Three percent of ammonium persulfate solution is prepared at
the time of use (see Note 3).
4. 10× running buffer (250 mM Tris–HCl, 1.92 M glycine, 1%
(w/v) SDS) is prepared by dissolving 30.3 g Tris, 144 g gly-
cine, and 10 g SDS in MQW and up to 1000 mL (see Note 4).
5. 4× sample buffer [124 mM Tris–HCl pH 7.5, 50% glycerol,
4% SDS, 0.2% bromophenol blue (BPB)] is prepared by mix-
ing 2 M Tris–HCl pH 7.5 with 1% BPB solution, glycerol,
SDS, and MQW (see Note 5).
6. Casein substrate solution 10 mg/mL is prepared from bovine
milk casein, which is dissolved in MQW with minimum amount
of 0.1 M NaOH, dispensed in small aliquots, and stored at
−20 °C until use (see Note 6).
7. Separation gel solution containing casein substrate (12.5%
acrylamide, 375 mM Tris–HCl pH 8.8, 0.1% SDS, and
200 μg/mL casein) is prepared by mixing 2 M Tris–HCl
pH 8.8 with 30% acrylamide stock solution and MQW, and the
mixture is degassed and then added with 10 mg/mL casein
substrate solution and 10% SDS (see Note 7).
8. Stacking gel solution (2.5% acrylamide, 125 mM Tris–HCl
pH 6.8, and 0.1% SDS) is prepared by mixing 0.5 M Tris–HCl
pH 6.8 with MQW and 10% acrylamide stock solution, and
the mixture is degassed and then added with 10% SDS.
2.2 Protease 1. Twenty-five percent of Triton X-100 stock solution is prepared

Activity Assay by dropwise adding 100% Triton X-100 into MQW with vig-
orous mixing (see Note 8).
2. Renaturation buffer (50 mM Tris–HCl pH 7.5, 2.5% Triton
X-100) is prepared by mixing 2 M Tris–HCl pH 7.5 with 25%
Triton X-100 stock solution and MQW. Prepare 200 mL buf-
fer per gel (see Note 9).
3. Calcium chloride stock solution 1 M is prepared by dissolving
powder CaCl2 in MQW.
4. Incubation buffer (50 mM Tris–HCl pH 7.5, 1 mM CaCl2) is
prepared by mixing 2 M Tris–HCl pH 7.5 with 1 M CaCl2 and
MQW. Prepare 200 mL buffer per gel (see Notes 10 and 11).
2.3 Reagents 1. Coomassie Brilliant Blue R-250 (CBB) stock solution for
for Detection RAMA stain (0.2% CBB and 60% methanol) is prepared as fol-
lows: First dissolve powder CBB R-250 in methanol, and after
complete dissolution, add MQW as to final concentration (see
Note 12).
2. Forty percent of ammonium sulfate stock solution for RAMA
stain is prepared by dissolving powder ammonium sulfate in
MQW.
3. Working solution of RAMA stain (0.05% CBB, 10% acetic acid,
15% methanol, 3% ammonium sulfate) should be freshly pre-
pared by mixing 0.2% CBB stock solution with acetic acid, 40%
ammonium sulfate stock solution, and MQW. Prepare 40 mL
per gel (see Note 13).
3 Methods
All procedures are carried out at room temperature.
3.1 Substrate Gel 1. For appropriate preparation of separation gel solution contain-
ing casein and SDS, order of mixing solutions and degassing are
important, as already mentioned in Subheading 2.1, item 7.
2. Add freshly prepared 3% ammonium persulfate and TEMED to
the degassed mixture of separation gel solution (see Note 14).
3. The resulting mixture is immediately poured into gel appara-
tus, covered with small amounts of MQW for avoiding air, and
incubated at room temperature until solidified (see Note 15).
3.2 Stacking Gel 1. As same as in Subheading 3.1, step 1, stacking gel also required
Preparation degassing for polymerization, and moreover, different from
and Setting Up separation gel, there is no air block off barrier of water.
2. Add freshly prepared 3% ammonium persulfate and TEMED
to the stacking gel solution.
3. The resulting mixture is immediately poured into gel appara-
tus and is inserted with a silicon comb for sample loading
(see Note 16).
4. Set up the electrophoresis apparatus.
5. Wash out any air bubbles from the bottom of gel and sample
lanes.
6. Flush sample lanes with electrode buffer by using small syringe
or pipette (see Note 17).
3.3 Sample 1. Mix protease samples with 4× sample buffer (see Note 18).
Preparation 2. Prepare a standard protease sample used for a positive control
(see Note 19).
3.4 Sample Loading 1. After preparation, immediately apply samples into sample lanes
(see Note 20).
2. Before loading, confirm the loading position of each sample in
a sample lane. Set a sample having large activity apart from the
others with two-lane space and not near by the small activity
(see Note 21).
3. When applied, load samples with smaller activity earlier. Do
not overflow any aliquots from respective sample lane, nor
push out any air bubbles into electrode buffer in a sample lane
(see Note 22).
3.5 Electrophoresis 1. Start electrophoresis without pre-run at constant current

10 mA per gel (see Notes 23 and 24).
3.6 Renaturation 1. Prepare renaturation buffer 200 mL per gel and pour into a
plastic vessel.
2. Wear plastic gloves, put the resolved gel into renaturation buf-
fer with shaking on a reciprocal shaker (see Note 25).
3. Incubate the gel on a reciprocal shaker for at least 40 min with
shaking at room temperature.
4. Discard renaturation buffer and add incubation buffer.
3.7 Incubation 1. After 10 min shaking in an incubation buffer, start incubation

at 37 °C (see Note 26).
2. After 18 h incubation, shake the gel on a reciprocal shaker at
room temperature for 10 min (see Notes 27 and 28).
3.8 Staining 1. Pour 40 mL freshly prepared working solution of RAMA stain
and Destaining per gel into another clean plastic vessel and put it on a recipro-
cal shaker (see Note 29).
2. Start shaking and transfer an 18 h-incubated gel into the stain
solution (see Note 30).
3. After 30 min stain with shaking, discard the solution and briefly
rinse with roughly 20 mL MQW two times (see Note 31).
4. Add fresh 200 mL MQW and incubate for 15 min with vigor-
ous shaking (see Note 32).
3.9 Scanning Image 1. Wear plastic glove, set a destained gel on an OHP sheet or
clear transparent plastic sheet avoiding any air bubbles under
the gel (see Note 33).
2. Set a gel with a sheet on a commercially available document
scanner (Epson GT X-970 or similar one) and scan it.
3. Process obtained images with PowerPoint and/or other
software for publication.
Fig. 1 Comparison of detection sensitivity among three methods such as conven-

tional, RAMA, and CGP casein zymography using trypsin as the test protease.
Purified bovine trypsin was separated on the casein-containing SDS-PAGE gels,
renatured, incubated at 37 °C, and protease activities were detected with (a)
conventional CBB stain, (b) RAMA stain, and (c) CGP stain. Trypsin was continu-
ously diluted from left to right by tenfold in each lane (starting from 100 ng to
1 pg) and applied on the gels at three different concentrations of the substrate
(casein), (a) 1 mg, (b) 200 μg, and (c) 67 μg. After separation, the gels were
incubated at 37 °C for 18 h. In (a), the gel was stained with conventional CBB
stain for 1 h and followed by an overnight destaining. In (b) and (c), gels were
stained and destained for total time of 1 h with RAMA and CGP stain, respectively.
All the stained gels were scanned at once, and their images were captured and
compared simultaneously
4. As example, the detection sensitivity among three zymography

methods such as conventional, RAMA, and CGP casein
zymography was compared, and trypsin was chosen. As shown
in Fig. 1, conventional method detected band at lane 4, RAMA
strongly at lane 5 and weakly but significantly at lane 6, and
CGP at lane 5. Therefore, sensitivity of RAMA casein zymog-
raphy was revealed to be tenfold superior to conventional one.
While CGP casein zymography demonstrated high sensitivity,
its contrast was poor. Therefore, RAMA casein zymography
was used for further experiments. In addition, the method was
much faster than conventional one.
5. To evaluate the applicability of this method to the other class
of protease, MMP7 was chosen. For MMP7, casein zymogra-
phy is known to be more suitable than gelatin one [22, 23]. As
shown in Fig. 2, conventional casein zymography faintly
detected the band at lane 3. On the other hand, RAMA casein
zymography significantly detected the band at lane 5.
Fig. 2 High sensitive detection of MMP7 (Matrilysin) by RAMA casein zymogra-

phy. Purified MMP7 was separated on the casein-containing SDS-PAGE gels,
renatured, incubated at 37 °C, and detected with (a) conventional CBB stain, (b)
RAMA stain. MMP7 was continuously diluted from left to right by tenfold in each
lane (starting from 105 ng to 1 pg) and applied on two different concentrations
of the substrate in the gels, (a) 1 mg, (b) 200 μg. After separation, the gels were
incubated at 37 °C for 18 h. In (a), the gel was stained with conventional CBB
stain for 1 h and followed by the overnight destaining. In (b), the gel was stained
with RAMA stain for 30 min and washed with water for 15 min. Both stained gels
were scanned at once, and their images were captured and compared
simultaneously
Therefore, RAMA casein was revealed to be more sensitive by

several tenfold than conventional one.
6. In conclusion, the method described in this chapter is simple,
rapid, reproducible, low-cost, needed non-expensive equip-
ments, and highly sensitive. Moreover, the strategy and method
described herein, the low substrate concentration coupled with
high sensitive staining, are also applicable to gelatin substrate
zymography for high sensitive detection of metalloproteinases.
Sensitivity is improved about tenfold (data not shown) [21].
4 Notes
1. Acrylamide and bisacrylamide are purchased at the highest

grade and the powders are stored at 4 °C in light shielding
bottles. To avoid wetting, do not open them until they reach
room temperature.
2. Since bisacrylamide is difficult to dissolve at high concentra-
tion, make sure that all the powders are dissolved thoroughly
and the solution is clear.
3. This solution should be prepared freshly and from power form,

otherwise actual percentage of prepared gel might vary day by
day. For storage of ammonium sulfate powder, temperature of
4 °C is not enough, because at that temperature it gradually
gets wet and decays. Therefore, the powder should be dis-
pensed into small aliquot in Eppendorf type tube with tight
cap and stored at −20 °C avoiding light until use. The dis-
solved solution should be discarded after use.
4. For high sensitive detection, all the equipments in contact with
this buffer including cylinders, bottles, stirrer bars, etc. should
be extremely clean. Since the powerful detergent of SDS is
contained in the solution at high concentration, it dissolves
residual proteases sticking to the vessel wall derived from bac-
teria, fungi, or biological specimens into the solution and their
activities are detected by zymography.
5. For sample handling and activity conservation, concentration
of Tris and glycerol and pH of the buffer are modified from
original report. To keep protease activity in the sample intact,
any reducing reagents are omitted.
6. Since casein does not dissolve in MQW at the concentration of
10 mg/mL, small amount of alkali is required to dissolve it.
However, too much alkali may disturb the separation dynamics
of electrophoresis. Although it is possible to prepare a 10 mg/
mL casein solution by using SDS instead of alkali, the sensitiv-
ity of SDS casein is slightly but significantly inferior to that of
alkali. When compared among several caseins and casein-like
proteins including skim milk and broking reagent, one from
Merck demonstrated the best results.
7. Oxygen contained in the solution is known to prevent acryl-
amide polymerization and to result in the weakness of the gel.
Therefore, degassing step is required for reproducibility of
actual gel hardness and of protease resolution on a gel. An
aspirator driven by running water is of low cost but unstable;
on the other hand, a vacuum pump is stable and preferable.
Since by the addition of SDS or casein into the solution, the
resulted mixture is very easy to make bubbles and the removal
of oxygen become insufficient, degassing should be performed
without SDS and casein.
8. When concentrated Triton X-100 is mixed with MQW, it
becomes solidified in the water and even under the vigorous
stirring it takes a long time to be dissolved. However, 25% solu-
tion is easy to mix with aqueous solution. To perform a whole
experiment in a short time, pre-preparation of 25% stock solu-
tion is required. To dissolve in a short time, it should be added
into water dropwise. Too much addition into water at once
results in stirrer bar stacking, and conversely, it sometimes
results in overnight stirring.
9. Previously, even in our publication [24], NaCl is contained in

a renaturation buffer as to final 0.1 M; however, it turned out
to be not required for protease activity and is omitted.
10. Final concentration of calcium at 10 mM is reported; however,
in my experiments it revealed to be rather inhibitory for the
activity comparing to 1 mM.
11. For trypsin activity, pH at 7.5 is not optimum and at 8.0 is
superior. However, former pH is suitable for many proteases
tested including serine- and metalloproteases except for aspar-
tic ones.
12. Since complete dissolving of CBB is required for high sensitiv-
ity and low background, powder CBB should be first dissolved
with only methanol in a clear transparent glass bottle and
stirred at least for 2 h. After confirmation by careful-visual-
inspection for complete dissolution (if not dissolved thor-
oughly, insoluble ones are observed as the small granulated
precipitates sticking on a vessel wall), the resulted dye solution
is then mixed with MQW and stock at room temperature.
13. Since working solution of RAMA stain is not so stable, it
should be used within several days after preparation. When
precipitates are found to exist in a solution, not only in a pre-
pared one, but also even in a fresh one, discard it and prepare
another one fresh. Complete dissolving of dye is required for
low background and uniform staining.
14. TEMED from several companies was tested. The one from
Sigma-Aldrich demonstrated the reproducible results.
15. Although for blocking off air from a surface of an acrylamide
solution isopropanol is also employed, it is inferior to water,
because of its bad smell and environmental pollution after use.
Since the relative density of gel solution is larger enough than
that of water, interface between gel solution and water is stable
against gentle addition of water. Pour water gently on it in a
small amount, not dropwise but continuously.
16. A silicon comb should be very clean, otherwise polymerization
of the gel surrounding comb results in incompletion. Several
times of insert actions and/or re-arrangements of comb posi-
tion might evoke air introduction into a solution and results in
incomplete polymerization and short gel forming. One action
of comb insertion and positioning is preferable for preparation
of good sample lanes.
17. Without washing, in the sample lanes unpolymerized acryl-
amide and gel debris remain and they perturbate sample load-
ing and separation on a gel.
18. Since this method is highly sensitive, most of the samples
should be diluted to a large extent to adjust the intensity of
protease band. Protein at a low concentration is unstable and

tends to stick to a vessel wall. Therefore, some manuals recom-
mend an addition of carrier protein for sample stability; how-
ever, commercially available low-cost bulk proteins are
sometimes contaminated with small amount of proteases, for
example, BSA with serum proteases. When analyzed, some-
times faint but significant bands appear. Therefore, simple
dilution with 4× sample buffer and immediate analysis are rec-
ommended. Once sample moves into substrate gel, it will be
surrounded by substrate proteins and detergent and might be
stabilized somehow.
19. Any sample containing known protease activity such as tissue
extracts, blood sample, or commercially available protease are
mixed together as the bulk, dispensed into small aliquots, and
stored at −20 °C. At time of use, one of the control samples is
mixed with 4× sample buffer and diluted to an appropriate
concentration and can be used for as a positive control, which
provides a reproducible activities on zymogram.
20. Since protein at a low concentration is time-dependently
adsorbed to vessel wall, even in the presence of SDS, prepared
samples should be loaded immediately.
21. Pre-experiments are required for rough estimation of the sam-
ple activities.
22. Since an air bubble spreads out small amounts of sample, if the
sample has large amounts of protease activity, entire sample
lanes might be contaminated by the protease activity. Therefore,
the lane without any proteases applied should be required as
the negative control.
23. In a previous publication, pre-run was recommended espe-
cially for casein zymography [25]. However, it took longer
time and the resolution of the bands was not so good. Because
interface between separation gel and stacking gel was partially
perturbed by pre-run, the sharpness and resolution were lost.
24. Apparatus size is 10.1 (W) × 8.2 (L) cm. At constant current,
quantity of heat produced during electrophoresis is constant,
and it keeps the temperature almost same and prevents excess
sample heating and loss of protease activity.
25. At first, put the resolved gel into renaturation buffer: do not
start handling the gel before renaturation buffer is ready. The
resolved gel tends to bend by itself and, by the contact with
each other, some amount of protease in a gel is transferred
even in a short time, and it results in the appearance of artificial
protease bands. However, when contacted each other in a buf-
fer under shaking conditions, such problems are almost
negligible.
26. Since the gel percent is relatively high (12.5%), the shaking
facilitates the equilibration of a gel with incubation buffer and
increases the reproducibility of the experiments.
27. Longer incubation does not always improve the band intensity,
partly because of its instability as a protease in a small amount.
28. By the shaking in a renaturation buffer, partially digested but
still remaining caseins in a protease zone are washed out from
gel before fixing and stained in a gel, and therefore, it enhances
the contrast between clear protease zone and stained
background.
29. Use one vessel for one gel.
30. For uniform staining, at the time of soaking a gel, shaking is
required.
31. The rinsing step is required for efficient washing.
32. Usually 15-min washing is enough to provide sufficient con-
trast. Although longer washing provides better contrast, too
much washing (3 h or more) may reduce the staining intensity
of undigested area and result in low contrast.
33. By using a plastic sheet, handling of the fragile gel on a scanner
such as changing orientation and position adjustment becomes
easier.
Acknowledgment
The author is grateful to Mariko Kawasaki-Yasumitsu for her excel-

lent support and also to members of Kamimachi Hospital. This
work was supported by a 2010 Incentive Grant from Yokohama
Academic Foundation.
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Hepatitis C virus directly acting antivirals: cur- physiological effects. Mycopathologia 171(5):299–
rent developments with NS3/4A HCV serine 323. doi:10.1007/s11046-010-9386-2
protease inhibitors. J Antimicrob Chemother 5. Antao CM, Malcata FX (2005) Plant serine pro-
65(10):2063–2069. doi:10.1093/jac/dkq284 teases: biochemical, physiological and molecular
2. Ruiz-Perez F, Nataro JP (2014) Bacterial ser- features. Plant Physiol Biochem 43(7):637–650.
ine proteases secreted by the autotransporter doi:10.1016/j.plaphy.2005.05.001
pathway: classification, specificity, and role in 6. Lu Q, Clemetson JM, Clemetson KJ (2005)
virulence. Cell Mol Life Sci 71(5):745–770. Snake venoms and haemostasis. J Thromb
doi:10.1007/s00018-013-1355-8 Haemost3(8):1791–1799.doi:10.1111/j.1538-
3. Raju RM, Goldberg AL, Rubin EJ (2012) 7836.2005.01358.x
Bacterial proteolytic complexes as therapeutic 7. Kini RM (2005) Serine proteases affecting
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venoms. Pathophysiol Haemost Thromb 18. SJFaZ W (1995) The catabolism of extracellu-
34(4–5):200–204. doi:10.1159/000092424 lar matrix. In: MAHaJR H (ed) Extracellular
8. Whitcomb DC, Lowe ME (2007) Human pan- matrix: a practical approach. Oxford University
creatic digestive enzymes. Dig Dis Sci 52(1):1– Press, New York, pp 261–287
17. doi:10.1007/s10620-006-9589-z 19. Yasumitsu H, Ozeki Y, Kawsar SM, Toda T,
9. Zasloff M (2002) Trypsin, for the defense. Nat Kanaly R (2010) CGP stain: an inexpensive,
Immunol 3(6):508–510. doi:10.1038/ odorless, rapid, sensitive, and in principle
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doi:10.1007/s00109-010-0677-3 (2010) RAMA stain: a fast, sensitive and less
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Chapter 3
Cysteine Protease Zymography: Brief Review

Jeff Wilkesman
Abstract
Cysteine proteases play multiple roles in basically all aspects of physiology and development. In plants, they
are involved in growth and development and in accumulation and mobilization of storage proteins.
Furthermore, they are engaged in signalling pathways and in the response to biotic and abiotic stresses. In
animals and also in humans, they are responsible for senescence and apoptosis, prohormone processing,
and ECM remodelling. When analyzed by zymography, the enzyme must be renaturated after SDS-
PAGE. SDS must be washed out and substituted by Triton X-100. Gels are then further incubated under
ideal conditions for activity detection. Cysteine proteases require an acidic pH (5.0–6.0) and a reducing
agent, usually DTT. When screening biological samples, there is generally no previous clue on what pepti-
dase class will be present, neither optimal proteolysis conditions are known. Hence, it is necessary to assess
several parameters, such as incubation time, pH, temperature, influence of ions or reducing agents, and
finally evaluate the inhibition profile. For detection of cysteine peptidase activity, the use of specific inhibi-
tors, such as E-64, can be used to prevent the development of cysteine peptidase activity bands and posi-
tively confirm its presence. Here four different protocols to assess cysteine protease activity from different
sources are presented.
Key words Cysteine protease, Zymography, Cathepsin
1 Introduction
Cysteine proteases were formerly known as thiol proteases. This

group of enzymes is characterized by having at its active site a thiol
group present in the Cys residue. Cysteine proteases have been
isolated from plants, animals, and bacteria. Among the most repre-
sentative examples are papain, actinidin, stem bromelain, ficin,
cathepsins B and H, streptococcal proteinases, clostripain, and cal-
pains. Typically, they all have relative small molecular masses, oscil-
lating between 20,000 and 35,000. Some Cys proteases are
glycosylated. The study of Cys proteases has become relevant,
especially for modification of food proteins and for synthesis of
biologically active peptides and their analogues.
The importance of cathepsins and other cysteine proteases
from viruses and parasites is that they are targets for key diseases
25
26 Jeff Wilkesman
such as cancer, AIDS, osteoporosis, arthritis, atherosclerosis, as

well as for parasitic diseases like amebiasis, leishmaniasis, malaria,
Chagas disease, and African sleeping sickness [1].
To distinguish cysteine protease activity from other proteases,
usually an inhibition assay must be performed in parallel. Typical
inhibitors are leupeptin and E-64 (Table 1) [2].
Recently published information [3] reveals that when cysteine
proteases—at least papain—are subjected to overlay zymography,
enzyme activity is frequently lost. This activity loss is associated
with the chemical lability of cysteine in the enzyme’s active site and
the presence of reactive oxidative species in the running buffer
when using electrophoresis under acidic conditions.
Multiplex cathepsin zymography has been reported [4] as an
effective assay based on SDS-PAGE, in order to quantify and
identify levels of active cathepsins K, L, S, and V in cells and tissue.
Among its benefits are: (a) antibodies are not required, hence the
method is inexpensive and species-independent, (b) staining of
the gel after non-reducing SDS-PAGE confirms cathepsin iden-
tity, (c) quantitative analysis is performed by densitometry, and
(d) multiplexed detection enables distinction of active cathepsins
K, L, S, and V in one cell or tissue extract, (e) cathepsin activity
quantification is performed in short timeframes.
Here, we present a very brief review of some general methods
to detect cysteine proteases.
Table 1
Some common inhibitors used for cysteine protease detection
Cysteine protease Optimal assay

inhibitor concentration Remarks
N-Ethylmaleimide Equimolar Binds stoichiometrically to SH groups. Prepare fresh stock
by dissolving 10 mg/mL water
Leupeptin 10–100 μM Inhibits calpain, cathepsin B, H, and L, and papain.
Prepare a 10 mM stock in water. Stock is stable for
6 months at −20 °C. Leupeptin also inhibits trypsin-
like serine proteases (e.g., trypsin, chymotrypsin,
pepsin, thrombin)
E-64 1–10 μM Will not inhibit serine protease (excepting trypsin).
A 1 mM stock is prepared in water and is stable at
−20 °C. Preparation in 50% ethanol has also been
reported [5]
Chymostatin 10–100 μM It is not specific as it inhibits also chymotrypsin-like
serine proteases as chymase cathepsins A, B, D, and
G. A 10 mM stock is prepared in DMSO and is stable
at −20 °C
Iodoacetic acid or 1–150 mM Covalent alkylation of the Cys residues at the active site.
iodoacetamide A 500 mM stock is freshly prepared in water
Cysteine Protease Zymography: Brief Review 27
2 Materials
2.1 Protocol 1: 1. 11% polyacrylamide gels containing 0.1% gelatine.

Cysteine Protease 2. 4% stacking gel.
Zymography
3. Sample buffer (2×): 50 mM Tris–HCl pH 6.8, 10% (v/v) glyc-
erol, 1% (w/v) SDS, 0.01% (w/v) bromophenol blue.
4. Activation buffer: 50 mM sodium citrate, 5 mM DTT, 5 mM
CaCl2, 1 mM ZnCl2, pH 5.0.
5. Staining solution: 0.05% Coomassie R-250, 10% acetic acid,
40% methanol.
6. Destaining solution: 10% acetic acid, 40% methanol solution.
2.2 Protocol 2: 1. Non-reducing loading buffer 5×: 0.05% bromophenol blue,

Cathepsin Zymography 10% SDS, 1.5 M Tris–HCl pH 6.8, 50% glycerol.
2. 12.5% SDS–polyacrylamide gels containing 0.2% gelatine.
3. 4.5% stacking gels.
4. Renaturing buffer: 65 mM Tris–HCl buffer pH 7.4 with 20%
glycerol.
5. Activity buffer: 0.1 M sodium phosphate buffer pH 6.0, 1 mM
EDTA. Add freshly 2 mM DTT just before use.
6. Coomassie stain solution: 10% (v/v) acetic acid, 25% (v/v)
2-propanol, 4.5% (w/v) Coomassie Blue R-250.
7. Destaining solution: 10% (v/v) 2-propanol, 10% (v/v) acetic
acid.
2.3 Protocol 3: 1. 11% polyacrylamide gels containing 0.1% gelatine.

General Cysteine 2. 4% stacking gel.
Zymography
3. 2.5% Triton X-100.
4. Developing buffer: (a) for acidic cysteine protease: 0.1 M
sodium citrate buffer pH 4.0, 0.05% Brij 35, 1 mM EDTA and
2 mM DTT; (b) for cysteine proteases with optimal neutral
pH: 0.1 M sodium phosphate buffer pH 6.8, 0.05% Brij 35,
1 mM EDTA and 2 mM DTT.
2.4 Protocol 4: 1. 12.5% SDS–polyacrylamide gels containing 0.2% gelatine.

Specific Cysteine 2. 4.5% stacking gels.
Zymography
3. 2.5% Tween 20.
Employing Inhibitors
4. Activation buffer: 50 mM acetic acid buffer pH 5.0; 2 mM
cysteine.
5. Staining solution: 0.5% amido black, 30% methanol, 10% acetic
acid.
6. Destaining solution: 30% methanol, 10% acetic acid.
28 Jeff Wilkesman
7. Inhibitors: final assay concentration prepared in activation buf-

fer. 20 mM EDTA, 25 mM pepstatin, 10 mM PMSF, and
10 μM E-64.
3 Methods
3.1 Protocol 1: 1. This protocol is based on the one proposed by Wagstaff et al.
Cysteine Protease [5], where proteins from petal tissue were analyzed.
Zymography 2. Prepare zymograms using 11% polyacrylamide gels containing
0.1% gelatin (type I from porcine skin) as substrate.
3. Use a 4% stacking gel as overlaid.
4. Load 5 μg protein per lane dissolved in sample buffer (2×).
5. Run gel under denaturing conditions at 180 V for 30 min.
6. The gels are renatured for 1 h in 2.5% Triton X-100.
7. Incubate gels overnight (15–18 h) at 37 °C in activation buffer.
If protease specificity is going to be tested, include an appro-
priate inhibitor in the activation buffer (see Notes 1 and 2).
8. Stain gels in staining solution.
9. Destain in destaining solution until areas of protease activity
are revealed as clear bands on a blue background.
10. Register the result by photographing or scanning.
3.2 Protocol 2: 1. This is a special case developed for cathepsin and based on
Cathepsin Zymography Wilder et al. [6] when analyzing recombinant cathepsins from
human sequences.
2. Add non-reducing loading buffer 5× to samples prior
loading.
3. Resolve equal amounts of cell or tissue protein in 12.5% SDS–
polyacrylamide gels containing 0.2% gelatin at 4 °C.
4. Remove gels carefully from the electrophoresis unit.
5. Renature enzymes in renaturing buffer. Repeat for a total of
three washes, 10 min each with gentle orbital shaking, at room
temperature (~22 °C).
6. Incubate gels in activity buffer for 30 min at room temperature
(~22 °C). It is possible to vary the pH value tested altering the
pH buffer or the chemical nature of the buffer (see Note 3).
7. Exchange activity buffer for fresh one (of the same pH) and
incubate further for 18–24 h (overnight) at 37 °C.
8. Discard activity buffer and rinse gels once with deionized water
and incubate for 1 h in Coomassie stain solution.
9. Destain in destaining solution.
3.3 Protocol 3: 1. Based on the method of Saitoh et al. [7], it is performed as a

General Cysteine usual SDS-PAGE.
Zymography 2. After SDS-PAGE, remove SDS by washing gel twice in 2.5%
Triton X-100 with gentle orbital shaking at room temperature
(~22 °C) for 15 min each.
3. Rinse gel briefly with distilled water twice.
4. Incubate gel at 37 °C in 15 ml developing buffer for 24 h
(see Note 4).
3.4 Protocol 4: 1. According to Grudkowska et al. [8], proceed to reactivate pro-

Specific Cysteine teins after electrophoretical separation by incubating gels twice
Zymography in 2.5% Tween 20 for 30 min each.
Employing Inhibitors 2. Rinse gels briefly three times with distilled water.
3. Incubate for 16 h (overnight) in 50 mM acetic buffer pH 5.0
containing 2 mM cysteine, with gentle shaking at 25 °C.
4. Rinse gels with water as before (step 2).
5. Stain for 3 h in staining solution [0.5% amido black, 30%
methanol, 10% acetic acid].
6. Destain gels in 30% methanol and 10% acetic acid, until clear
bands are visible on a dark blue background.
7. To detect proteinase specificity, use inhibitors. Prepare inhibi-
tors to the indicated assay concentration: 20 mM EDTA (for
metalloproteinases), 25 mM pepstatin (for aspartic protein-
ases), 10 mM PMSF (for serine proteinases), and 10 μM E-64
for inhibition of cysteine proteinases [9]. Other inhibitors may
be used (see Table 1).
8. Incubate protein extracts for 2 h in 50 mM acetic buffer
pH 5, containing the appropriate inhibitor, prior to
electrophoresis.
9. After electrophoreses, place the gels (or gel strips if individual
lanes are going to be tested) in the presence of the inhibitor.
Gelatin digestion must be carried out in the presence of the
inhibitors.
10. Again, incubate for 16 h (overnight) in 50 mM acetic buffer
pH 5.0 containing 2 mM cysteine plus the inhibitor, with gen-
tle shaking at 25 °C.
11. Rinse gels with water as before (step 2).
12. Stain for 3 h in staining solution [0.5% amido black, 30%
methanol, 10% acetic acid].
13. Contrast the results. The presence of the specific inhibitor will
diminish the intensity of the band signal corresponding to cys-
teine protease (see Notes 5–7).
30 Jeff Wilkesman
4 Notes
1. In order to determine the specificity of the bands correspond-

ing to cysteine protease, a protease inhibitor must be used. For
this purpose, include in the overnight activation buffer either
(a) 2 μM leupeptin or (b) 100 μM E-64 in 50% ethanol. Always
perform a control without inhibitor. Alternatively, IAA can be
added in the sample buffer before submitting the sample to
electrophoresis. As this is covalent modification, there should
be no need to add more IAM at the activation buffer. However,
tests should be run before to check the inhibitor efficiency.
2. The use of other inhibitors is also recommended, e.g., PMSF,
EDTA, and pepstatin, in order to rule out any other concomi-
tant protease with similar Rf.
3. For different pH conditions, use, e.g., 0.1 M sodium acetate
buffer pH 4, or sodium phosphate buffer pH 6, 7or 8.
However, be aware that if using phosphate buffer, calcium and
magnesium ions present will precipitate in the respective phos-
phate salts.
4. Take into consideration that for acidic cysteine protease, use
0.1 M sodium citrate buffer pH 4.0, but for cysteine proteases
with an optimal neutral pH, use 0.1 M sodium phosphate buf-
fer pH 6.8. Other buffers may also be employed.
5. Care must be taken when analyzing inhibition results. Some
cysteine proteases may present unspecific inhibition towards
other type of inhibitors.
6. Take into consideration that some specific cysteine protease
inhibitors are dissolved in solvents other than water, like
DMSO or ethanol. A control must be run to interpret cor-
rectly the result, as it may turn that the solvent alone may
inhibit the enzyme as well.
7. Quantification of the inhibition process can be performed via
densitometry. A calibration curve must be previously gener-
ated employing a commercial standard cysteine protease, with
and without the presence of the inhibitor. Intensity of band is
proportional to the activity. It is suggested to test with differ-
ent enzyme concentrations and different inhibitor concentra-
tion until optimal band signals intensities are achieved on the
gel. For this purpose, staining must be first standardized.
Acknowledgments
The author thanks Dr. Rebeca Giamate and Dr. Liliana Kurz from
the University of Carabobo for technical and emotional support
during the writing and editing of this chapter.
References
1. Verma S, Dixit R, Pandey KC (2016) Cysteine lytic activity during senescence of Alstroemeria
proteases: modes of activation and future pros- petals. J Exp Bot 53:233–240
pects as pharmacological targets. Front 6. Wilder CL, Park KY, Keegan PM, Platt MO
Pharmacol 7:107 (2011) Manipulating substrate and pH in
2. Smith C, Gates R. Protease inhibition and zymography protocols selectively distinguishes
detection. Life Science. Biofiles 4(2). http:// cathepsins K, L, S, and V activity in cells and
www.sigmaaldrich.com/content/dam/sigma- tissues. Arch Biochem Biophys 516:52–57
aldrich/flashapps/biofiles-movie/pdf/ 7. Saitoh E, Yamamoto S, Okamoto E, Hayakawa Y,
BioFiles_4.2_v1.pdf. Accessed 27 Sep 2016 Hoshino T, Sato R, Isemura S, Ohtsubo S,
3. Vootukuri Reddy S, Philpott MP, Trigiante G Taniguchi M (2007) Identification of cysteine
(2016) Retaining in-gel zymographic activity of proteases and screening of cysteine protease inhib-
cysteine proteases via a cysteine-supplemented itors in biological samples by a two-dimensional
running buffer. Electrophoresis 37:2644–2648 gel system of zymography and reverse zymogra-
4. Dumas JE, Platt MO (2013) Systematic optimi- phy. Anal Chem Insights 2:51–59
zation of multiplex zymography protocol to 8. Grudkowska M, Lisik P, Rybka K (2013) Two-
detect active cathepsins K, L, S, and V in healthy dimensional zymography in detection of pro-
and diseased tissue: compromise among limits teolytic enzymes in wheat leaves. Acta Physiol
of detection, reduced time, and resources. Mol Plant 35:3477–3482
Biotechnol 54:1038–1047 9. Zhang N, Jones BL (1995) Characterization of
5. Wagstaff C, Leverentz MK, Griffiths G, Thomas germinated barley endoproteolytic enzymes by
B, Chanasut U, Stead AD, Rogers HJ (2002) two-dimensional gel electrophoresis. J Cereal
Cysteine protease gene expression and proteo- Sci 21:145–153
Chapter 4
Aspartic Protease Zymography Case Study: Detection

of Fungal Acid Proteases by Zymography
Gavin Kernaghan and Michael Mayerhofer
Abstract
This chapter describes a method for the production and characterization of fungal acid proteases. Protease
production is induced by growth on BSA media over a pH gradient and protein levels are monitored over
time with the Bradford assay. Once protein is depleted, the media is purified and proteases are character-
ized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below
those found in traditional zymographic systems avoids the potential loss of activity that may occur in
aspartic proteases under alkaline conditions.
Key words Neutral zymography, Fungal enzymes, Protein utilization, SDS-PAGE, Laemmli gels,
Bradford assay
1 Introduction
Fungi exhibit external digestion and therefore rely heavily on

extracellular enzymes to access nutrient resources. Fungal cells
secrete a wide variety of extracellular proteases for the degradation
of proteins, including aspartic, cysteine, serine, and metallo-
proteases [1–3]. Both the production and activity of proteases are
optimal at specific pH values, which vary among protease types and
fungal species [4].
Zymography is a useful tool for characterizing extracellular
proteases, as it allows for direct visualization of enzymatic activity.
Standard zymography utilizes SDS-PAGE with the Laemmli buff-
ering system [5]. This involves basic Tris-glycine running buffer
and basic Tris-chloride gel buffer, resulting in pH levels up to 9.5
[6]. However, many fungal proteases (e.g., aspartic proteases)
exhibit optimal activity at low pH (as low as 2.0) [7] and can
become unstable and susceptible to structural modification or
irreparable inactivation under the alkaline conditions found within
a Laemmli gel [8, 9]. Therefore, a neutral to acidic environment
should be maintained in order to retain the enzymatic activity of
33
34 Gavin Kernaghan and Michael Mayerhofer
aspartic proteases. Although this has been achieved through the

addition of compounds such as potassium acetate [10], this results
in a system that may be acidic, but not buffered at the desired pH,
and therefore subject to fluctuation.
More recently, a modification to protein electrophoresis has been
developed in which the standard Tris-glycine gel buffer is replaced
with bis-Tris and the standard Tris-chloride running buffer is replaced
with MES-Tris or MOPS-Tris [11]. This results in a near-neutral pH
system and represents a general improvement in protein electropho-
resis as resolution is increased through reductions in protein–acryl-
amide interactions and disulfide formation. The neutral system also
reduces the breakdown of buffer components and increases gel shelf
life. This system is easily adapted to zymography, reducing the prob-
lems related to aspartic proteases under alkaline conditions.
Here we describe a method in which near-neutral electropho-
resis is adapted for the zymography of fungal aspartic proteases.
The method involves the induction of fungal proteases and subse-
quent analysis by zymography under near-neutral conditions.
Cultures are grown on protein media (BSA) over a range of pH
levels until the protein is exhausted (based on Bradford assay).
Media is then purified and analyzed using zymography with gels
and buffers at near-neutral pH.
2 Materials
2.1 Acid Protease 1. Fungal cultures: obtain fungal isolates from culture collections
Production or by direct isolation using media appropriate for the fungal
group (see Note 1).
2. Water agar medium: 1.5% agar in distilled H2O (dH2O).
3. BSA medium (1 L): 0.3 g BSA (96% lyophilized powder,
Sigma), 0.03 g yeast extract, 1 g MgSO4·7H2O, 2 g KH2PO4,
1 mg FeSO4, 1 mg ZnSO4·7H2O, 0.1 mg MnSO4·H2O,
0.1 mg H3BO3, 0.1 mg CoCl2·6H2O, 0.1 mg CuSO4·5H2O
(see Note 2).
4. Basic lab material: Petri dishes, stericup filter units (0.22 μm
filter), automatic pipetter with a 0.22 μm filter, sterile 1 and
10 mL pipettes, sterilized 18 × 150 mm culture tubes, loose
fitting culture tube closures (e.g., Kap-Uts from Bellco), clear,
flat-bottom 96-well microplates.
5. Laminar flow hood or biosafety cabinet.
6. Incubator for fungal cultures.
7. Transfer tubes (Spectrum Labs).
8. Microplate reader (spectrophotometer) capable of reading
absorbance at 580 nm.
9. Bradford assay reagent (Coomassie Brilliant Blue-G reagent).
Zymography of Fungal Acid Proteases 35
2.2 Near-Neutral 1. Centrifuge capable of spinning 15 mL tubes at 18,400 × g and

Zymography 4 °C.
2. Centrifugal filters with 10 kDa cutoff (e.g., Microsep or
Amicon).
3. Mini-protean protein electrophoresis cell (BioRad) and power
supply.
4. Gelatin solution: 1% Gelatin in dH2O.
5. Resolving gel: 2.05 mL dH2O, 2.85 mL 1.25 M bis-Tris buffer
(pH 6.7), 4 mL 30% acrylamide:bis-acrylamide (29:1), 100 μL
10% sodium dodecyl sulfate (SDS) in dH2O, 1 mL 1% gelatin
in dH2O, 20 μL N,N,N′,N′-tetramethyl-ethylenediamine
(TEMED), 7.5 mg ammonium persulfate (APS).
6. Stacking gel: 2.88 mL dH2O, 1.42 mL 1.25 M bis-Tris buffer
(pH 6.7), 0.65 mL 30% acrylamide:bis-acrylamide (29:1),
50 μL 10% SDS in dH2O, 25 μL TEMED, 7.5 mg APS.
7. Running buffer: 250 mM 3-(N-morpholino)propanesulfonic
acid (MOPS), 250 mM Tris(hydroxymethyl)aminomethane
(Tris), 0.1% SDS in dH2O, stored at 4 °C.
8. Loading buffer: 125 mM Tris–HCl (pH 6.8), 8% SDS, 50%
glycerol, 0.02% bromophenol blue.
9. 10–250 kDa protein standard (Precision Plus; Bio Rad).
10. Wash buffer: 2.5% Triton X-100.
11. Development buffer: 200 mM NaCl, 5 mM CaCl2, 0.02% Brij
L23 solution, in 50 mM sodium citrate–citric acid buffer.
12. Staining Solution: 45% methanol, 10% glacial acetic acid, 45%
dH2O, 0.1% amido black.
13. Destaining solution: 50% methanol, 2% glacial acetic acid, 48%
dH2O.
14. Flatbed scanner (300–600 dpi).
3 Methods
3.1 Growth of Fungal 1. Plate isolates onto water agar medium (see Note 3).
Cultures 2. Allow cultures to grow to a point at which there is enough
and Optimization mycelium to transfer. The optimum growth temperature will
of Protease Utilization depend on the group of fungi under investigation.
3. Autoclave empty culture tubes for 15 min at 121 °C. Enough
tubes are required to accommodate all replicates of each isolate
at each pH level of interest (see Note 4). Label each tube with
respect to isolate, replicate, and pH.
4. Prepare liquid BSA media (see Notes 5 and 6).
5. Set up a range of media pH levels by dividing medium into

batches and titrating each batch to the desired pH with 0.1 M
HCl or NaOH (see Note 7).
6. Filter-sterilize each batch of pH-adjusted BSA medium with
0.22 μm filters using a glass vacuum filter holder or disposable
Stericup filter units (see Notes 8 and 9).
7. Using sterile pipettes and an automatic pipetter with a 0.22 μm
filter, add 10 mL of each pH-adjusted media to culture tubes
labeled with respect to fungal isolate, pH level, and replicate
number. Attach loose fitting closures to culture tubes. If quan-
tification of protein utilization is desired, retain a small amount
of each batch (media at each pH level) for constructing stan-
dard curves in step 11.
8. In a laminar flow hood or biosafety cabinet, use sterile
Transfertubes to transfer each fungal isolate from water agar to
the corresponding culture tubes containing pH-adjusted liq-
uid BSA media.
9. Test the liquid cultures for protein content by removing a
small aliquot (e.g., 0.1 mL) of BSA media from each culture
using sterile pipettes and a filtered pipetter in a laminar flow
hood or biosafety cabinet.
10. Add 6 μL of each aliquot to each of three wells of a clear flat-
bottom 96-well microplate (see Note 10). Also add 6 μL dH2O
to each of three wells to assess background absorbance levels.
11. If protein quantification is desired, prepare a protein standard
curve using the fresh BSA media retained at step 8 (see Note 11).
12. Add 56 μL Coomassie protein assay reagent (see Note 12).
13. Incubate plate at room temperature for 15 min.
14. Place plate in spectrometer and read absorbance at 580 nm.
15. Repeat the Bradford assay on a regular basis (see Note 13)
until it indicates that the BSA has been completely utilized.
Zymograms are run when two or more consecutive Bradford
readings are not significantly above the background spectrom-
eter readings (see Notes 14 and 15).
3.2 Near-Neutral 1. Filter the fungal hyphae from the BSA media using a coarse
Zymography filter. Sterile conditions are not necessary (see Note 16).
2. Centrifuge 7.5 mL of each filtered media sample for 10 min at
18,400 × g in order to remove residual fragments of fungal
hyphae.
3. Concentrate 5 mL of the resulting supernatant with centrifu-
gal filters with a 10 kDa cutoff at 7600 × g for 20 min at 4 °C.
4. Keep filtrate in the refrigerator or on ice until use in
zymography.
5. Set up glass gel plates for electrophoresis cell.

6. Prepare a fresh 1% gelatin solution (5 mL).
7. Mix components of the stacking and resolving gels separately,
leaving out APS and TEMED until just prior to pouring gel
(see Note 17).
8. Dissolve 7.5 mg APS and 20 μL TEMED in the resolving gel
mixture.
9. Pipette 3–3.5 mL of the liquid resolving gel between the gel
plates (see Note 18).
10. Pipette a layer of 70% ethanol on top of the resolving gel
(see Note 19).
11. Let polymerize (approximately 1 h) and blot excess ethanol.
12. Dissolve 7.5 mg APS and 25 μL TEMED in the stacking gel
mixture.
13. Pipette the stacking gel mixture between the glass plates until
full.
14. Add an 8- or 12-well comb and allow to polymerize (approxi-
mately 30 min).
15. Place completed gel plates into electrophoresis cell sitting in an
ice bath.
16. Add cold running buffer.
17. Gently remove comb (see Note 20).
18. Pipette a small amount of running buffer in and out of the
wells to remove any interfering gel material.
19. Mix each sample 3:1 with 4× loading buffer (see Note 21).
20. Load the protein standards (10 μL) and samples (see Note 22).
21. Run gel at 125 volts until the dye front is almost at the bot-
tom of the gel and the protein standards are clearly separated
(see Note 23).
22. Remove the gel plates from electrophoresis box and separate
them carefully (see Note 24).
23. Wash three times in 2.5% Triton X-100 for 15 min each time.
24. Wash in development solution for 15 min (see Note 25).
25. Incubate in development solution overnight at 37 °C.
26. Transfer the gel from the development buffer to the staining
solution for 40–60 min and shake gently at room
temperature.
27. Remove and place in the destaining solution for 15 min.
28. Repeat this step until bands of clearing become visible (Fig. 1).
29. Scan gel using a flatbed scanner at 300–600 dpi.
Fig. 1 Examples of fungal aspartic proteases from four fungi characterized by

near-neutral zymography. (a) Irpex lacteus (42 kDa); (b) Meliniomyces variabilis
(60 and 20 kDa); (c) Phialocephala fortinii (63 and 26 kDa); (d) Umbelopsis isa-
bellina 70 kDa. Each zymogram includes a 10–250 kDa protein standard
30. Analyze gel images for protease size (kDa) and intensity using
software such as Gel-Pro Analyzer or Image J.
31. To confirm the aspartic nature of the proteases on the gel, the
specific inhibitor Pepstatin A (1 mM) can be incubated with
the sample prior to loading on the gel, or added to the devel-
opment solution after running the gel, or both. Loss of indi-
vidual bands after the addition of Pepstatin A indicates an
aspartic protease (see Note 26).
4 Notes
1. Most culturable filamentous fungi can be maintained on stan-

dard malt or yeast extract–malt extract agar (YMA) plates.
Many yeasts can be maintained on yeast extract peptone dex-
trose (YPD) plates.
2. For convenience, the solutions of FeSO4, MnSO4·H2O,
H3BO3, ZnSO4·7H2O, CoCl2·6H2O, and CuSO4·5H2O can
be kept as a 1000× nutrient stock solution, and added to the
basal media at 1 mL/L.
3. Passage through water agar avoids carryover of nutrients from
rich media such as YMA. Ideally, all Petri dishes should contain
the same amount of water agar in order to reduce weighing
errors if biomass determination after growth in liquid BSA is
desired. We fill 9 mm diameter Petri dishes with 25 mL media.
4. We grow the fungi over a range of pH values to optimize growth
and protease production as only small amounts of protease may
be produced under conditions of suboptimal pH. For example, if

no prior information is available on pH optima for the fungi of
interest, each isolate could be grown at pH 2–7 (in one unit
increments).
5. For each isolate, 10 mL of medium is required for each repli-
cate culture at each pH level. For example, each isolate would
require 180 mL given three replicates, each at pH 2–7. As we
also use a small amount of media for creating a standard curve
at each pH, we would prepare at least 250 mL for each fungal
isolate.
6. Add BSA in small amounts to avoid clumping.
7. We found it most effective to work with the largest volume of
media possible. After initially determining the pH of the bulk
media, titrate it all to the closest desired pH value, then split
the solution as necessary and titrate each part up or/and down
to achieve the desired pH range.
8. Filter sterilization should be done in a laminar flow hood or
biosafety cabinet to avoid contamination.
9. We find that filter sterilizing the complete media is quite effi-
cient and also avoids any potential pH changes caused by auto-
claving. However, it may be more economical to autoclave the
media prior to adding the BSA and yeast extract, and then
adding these to the cooled media using a 0.22 μm syringe
filter.
10. We are describing the Bradford assay developed for microplate
readers. A single cuvette spectrometer could also be used, but
would be time-consuming. The 96-well format also lends itself
well to make each measurement in triplicate, allowing outlier
readings (often caused by bubbles in Coomassie reagent) to be
discarded.
11. The Bradford assay is somewhat pH-sensitive, so it is best to
create protein standard curves using fresh media at the same
pH as the growth media being tested. To prepare a standard
curve, pipette 12 μL of media into the first well. Then add 6 μL
of distilled water to wells 2–10. Then transfer and mix 6 μL
from well 1 to well 2, and 6 μL from well 2 to well 3. Continue
until well 10, from which 6 μL is removed and discarded.
12. Pipette Coomassie protein assay reagent directly on top of the
samples of media. Do not mix, as this may create bubbles that
may interfere with the absorbance readings.
13. The time interval for sequential measurement of the media
protein depends on the fungi being tested. At optimal pH,
some fungi may completely deplete the protein very rapidly
and should be measured every day or two. Other fungi may
take 2 weeks or more.
14. At the point at which the BSA has been exhausted, it no longer
has the potential to interfere with the enzymes on the zymo-
grams. The rate of protein loss also indicates which cultures
(pH levels) are producing the highest levels of protease and are
therefore most likely to result in successful zymograms. We
find it effective to graph protein loss over time in order to
determine which cultures are most active, and when to assay
proteases.
15. Once the optimal pH for protease production has been deter-
mined, isolates could optionally be grown on 1% BSA media at
that pH. The higher concentration of BSA induces more pro-
tease production and results in greater clearing of the protein
background during zymography, although it will take longer
for the cultures to exhaust the BSA.
16. We find that organza fabric works well as a coarse filter. Fungal
biomass can also be dried and weighed at this point if data on
biomass is desired.
17. The recipe given is for two gels. If only one is required, the
other can be wrapped in plastic and stored in the refrigerator.
18. From 3 to 3.5 mL of the resolving gel mixture should be about
6 cm high between the glass plates, leaving space for the stack-
ing gel.
19. Ethanol, distilled water, 0.1% SDS, or butanol can be added to
obtain a flat surface at the top of the resolving gel.
20. Gently remove the comb by wiggling as the stacking gel tends
to stick to it.
21. A 4× sample buffer helps to maximize the amount of sample
loaded in the well. For example, 75 μL of sample plus 25 μL of
loading buffer will be enough to load two 50 μL wells.
22. We recommend loading two wells of each sample to maximize
the chance of bands with good resolution.
23. If only high molecular weight proteases are present, the gel
could be run until the first or second bands of the protein stan-
dard are run off the gel.
24. The flat end of a scientific spatula works well for prying apart
the plates.
25. The pH of the development solution can be modified in order
to determine the pH optimum and range of individual prote-
ases based on band intensity. The pH of the development solu-
tion can be brought as low as 2.5 by adjusting the ratio of
sodium citrate to citric acid.
26. Similarly, the specific inhibitors E-64 (1 mM), PMSF (1 mM),
and EDTA (10 mM) can be used to indicate serine proteases,
cysteine proteases, or metallo-proteases, respectively.
References
1. Pavlukova EB, Belozersky MA, Dunaevsky YE 7. Leake JR, Read DJ (1990) Proteinase activity
(1998) Extracellular proteolytic enzymes of in mycorrhizal fungi. I: The effect of extracel-
filamentous fungi. Biochemistry (Mosc) 63: lular pH on the production and activity of pro-
899–928 teinase by ericoid endophytes from soils of
2. Monod M, Capoccia S, Léchenne B et al contrasted pH. New Phytol 115:243–250
(2002) Secreted proteases from pathogenic 8. Piper DW, Fenton BH (1965) pH stability and
fungi. Int J Med Microbiol 292:405–419 activity curves of pepsin with special reference
3. Yike I (2011) Fungal proteases and their patho- to their clinical importance. Gut 6:506
physiological effects. Mycopathologia 171: 9. Kulkarni A, Gaikwad S, Rao M (2008) pH
299–323 induced structural alterations in an aspartic
4. Mayerhofer MS, Fraser E, Kernaghan G (2015) protease from Vigna radiata indicating an
Acid protease production in fungal root endo- alkali induced molten globule state. Int J Biol
phytes. Mycologia 107:1–11 Macromol 43:373–376
5. Laemmli UK (1970) Cleavage of structural 10. Burton KS, Smith JF, Wood DA, Thurston CF
proteins during the assembly of the head of (1997) Extracellular proteinases from the
bacteriophage T4. Nature 227:680–685 mycelium of the cultivated mushroom Agaricus
6. Hachmann JP, Amshey JW (2005) Models of bisporus. Mycol Res 101:1341–1347
protein modification in Tris–glycine and neu- 11. Updyke TV, Engelhorn SC (2000) System for
tral pH Bis-Tris gels during electrophoresis: pH-neutral stable electrophoresis gel. US
effect of gel pH. Anal Biochem 342:237–245 patent 6162338 A
Chapter 5
Detection of Aspartic Proteinase Activities Using Gel

Zymography
Handunge Kumudu Irani Perera
Abstract
Gel zymography is a two-stage process where the proteins from the test sample are first separated by elec-
trophoresis followed by the detection of the activity of hydrolytic enzymes. Many zymography procedures
use sodium dodecyl sulfate (SDS) polyacrylamide gels copolymerized with an appropriate substrate. The
procedure described here uses native polyacrylamide gel electrophoresis (PAGE) in the absence of both
SDS and substrate. In order to visualize aspartic proteinase activity, the gel is impregnated in bovine hemo-
globin at pH 3.0 for 15 min after the electrophoresis procedure. Subsequently, the gel is incubated in a
humid container in the absence of hemoglobin for 1 h at 37 °C. At the end, the gel is stained with amido
black and destained. Clear areas against a dark background corresponding to aspartic proteinase activities
can be detected.
Key words PAGE, Zymography, Aspartic proteinases, Hemoglobin
1 Introduction
Gel zymography is a two-stage process where the proteins from the

test sample are first separated by electrophoresis and then the activ-
ity of hydrolytic enzymes is detected. Qualitative or quantitative
information on hydrolytic enzymes [1] is collected based on the
visualization of the breakdown of a substrate [2]. Zymography is
mostly used to detect the activities of the proteolytic enzymes [3].
It is a method used for screening, identification, and characteriza-
tion of proteinases [4]. Rather than visualizing all proteins sepa-
rated on the gel using a general staining method, zymography
recognizes a particular group of enzymes which remain active after
the electrophoresis. Zymography is a flexible procedure which pro-
vides room for the investigator to change the substrate, pH, tem-
perature, ion concentration as well as to incorporate inhibitors
according to the type of enzyme which is under investigation [1].
The method elaborated here describes a gel zymography pro-
cedure designed to recognize activities of aspartic proteinases
43
44 Handunge Kumudu Irani Perera
which belong to one catalytic class of proteinases [4]. One specific

feature of the aspartic proteinases is having acidic pH optima. They
are usually inactive at neutral pH, unlike most other enzymes.
Many zymography procedures use sodium dodecyl sulfate
(SDS) polyacrylamide gel electrophoresis which separates proteins
based on their molecular weight [4, 5]. The procedure described
here uses native polyacrylamide gel electrophoresis (PAGE) in the
absence of SDS. Native polyacrylamide gel electrophoresis (PAGE)
separates proteins in their native conformation under non-
denaturing and non-reducing conditions [6]. A key determinant of
the protein separation by PAGE is the net charge of the protein as
governed by its amino acid composition and pH of the medium
during electrophoresis [6]. Advantage of preserving the active
state during separation of a protein with the native PAGE method
is used in the described procedure to demonstrate the proteinase
activity. PAGE method can resolve proteins of the same molecular
mass as long as their net charges are considerably different [7].
Generally, zymography procedures use resolving gels copoly-
merized with a substrate [4, 5]. Gels copolymerized with gelatin
have long been used to detect matrix metalloproteinase activities
[1]. In the present technique, the substrate solution at the opti-
mum pH was added to the gel after the protein separation is com-
pleted. Hemoglobin, which is the most common substrate used to
measure aspartic proteinase activity in biological samples [8], is
used in this procedure. After allowing time to react with the sub-
strate, the gel is stained with amido black and destained [9]. The
proteinase activities can be seen as clear areas against a dark back-
ground due to the breakdown of protein substrate [1]. Activity
due to multiple enzymes can be detected using this procedure as
they will be visualized separately.
2 Materials
2.1 Polyacrylamide 1. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Weigh 45.43 g
Gel of Tris and add into a 250 mL graduated beaker. Add 200 mL
distilled water and mix the contents using a stir bar. Adjust the
pH to 8.8 using HCl (see Note 1). Make the volume up to
250 mL with distilled water. Store at 4 °C.
2. Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8. Weigh 15.14 g
Tris and add into a 250 mL graduated beaker. Add 200 mL
distilled water and mix the contents using a stir bar. Adjust the
pH to 6.8 using HCl (see Note 1). Make the volume up to
250 mL with distilled water. Store at 4 °C.
3. Acrylamide (30%), bisacrylamide solution: Acrylamide 29.2%,
bisacrylamide 0.8%. Weigh 73 g of acrylamide and 2 g
bisacrylamide into a beaker containing 100 mL of distilled
Aspartic Proteinase Zymography 45
water (see Note 2). Mix and make up to 250 mL with dis-
tilled water. Filter and store at 4 °C in a dark bottle.
4. 10% ammonium persulfate (APS): 10% APS. Weigh 100 mg of
ammonium persulfate in a microfuge tube and add 1 mL of
distilled water. Mix and store at 4 °C after covering with alumi-
num foil (see Note 3).
5. Tetramethylethylenediamine (TEMED): Store at 4 °C.
6. PAGE running buffer (×10): 250 mM Tris, 1.92 M glycine.
Weigh 30 g Tris, 144 g glycine and dissolve in 1 L distilled
water (see Note 4).
7. PAGE running buffer (×1): 25 mM Tris, 192 mM glycine.
Add 130 mL of running buffer (×10) to distilled water and
make up to 1300 mL (see Note 4). Store at 4 °C.
8. PAGE sample buffer (×3): 0.5 M Tris–HCl (pH 6.8)
(93.75 mM), glycerol (37.5%), and bromophenol blue
(0.015%). Add 187.5 μL 0.5 M Tris–HCl (pH 6.8), 375 μL
glycerol, and 100 μL of 0.15% (150 mg in 100 mL distilled
water) bromophenol blue into a microfuge tube. Add distilled
water up to 1 mL. Store at 4 °C.
2.2 Zymography 1. Formate buffer: 100 mM formate buffer. Add 5.75 mL formic
Components acid (80% solution) into 900 mL distilled water. Adjust to
pH 3.0 with concentrated NaOH solution. Dilute up to 1 L
with distilled water. Store at 4 °C.
2. Bovine hemoglobin solution (Hb): 1% bovine Hb, pH 3.0. Prepare
fresh (20 mL/gel). Add 200 mg Hb in 20 mL distilled water. Mix
with a magnetic stirrer for about 1 h. Check the pH using a pH
paper and get it to pH 3.0 by adding HCl drops (see Note 5).
3. Amido black stain: 0.1% Amido black, 10% acetic acid, 30%
methanol. Weigh 100 mg Amido black 10 B. Add 7 mL acetic
acid and 30 mL methanol. Make up to 100 mL in distilled
water, mix with a magnetic stirrer to dissolve. Store at room
temperature.
4. Destain: Methanol 20 mL (20%), glacial acetic acid 10 mL
(10%), and distilled water up to 100 mL (70%). Store at room
temperature.
3 Methods
It is extremely important to minimize the loss of activity of

enzymes until the zymography is done (see Notes 6–9).
Furthermore, having an idea about the optimum pH, activity of
the enzyme, and whether the protein is negatively charged at
pH 8.5 will facilitate the selection of optimum conditions for
zymography (see Notes 10 and 11).
3.1 Preparation 1. Ovaries of pigs (6–8 months age) are obtained from an abattoir
and Fractionation during routine operations. Ovaries are cleaned and brought to
of Porcine Ovarian the laboratory in dry ice. All procedures are conducted at 4 °C.
Extracts 2. Crude ovarian extract is obtained by homogenizing the ovaries
in 50 mM phosphate-buffered saline, pH 7.5. Extract is centri-
fuged to remove debris and the supernatant is dialyzed over-
night in 20 mM phosphate buffer.
3. This extract is centrifuged again at 15,000 × g.
4. Supernatant is fractionated using DEAE-52 cellulose equili-
brated with 20 mM Tris–HCl (pH 8.5). The bound proteins
are eluted with a 0–1 M linear NaCl gradient.
5. Active fractions are subjected to Sephacryl S-200 column,
equilibrated with 20 mM phosphate buffer pH 7.5, containing
0.2 M NaCl. Crude extract and the partially purified fractions
are then subjected to gel zymography.
3.2 Polyacrylamide 1. Standard protocol of PAGE is used [9–12]. Single percentage

Gel Electrophoresis (7.5%) 1 mm thickness mini gels are casted. Tris glycine buffer
Under Non-Denaturing system is used.
Conditions 2. Assemble the gel cassette according to the manufacturer’s rec-
ommendations (see Note 12). Place the comb into the cassette.
Using a marker pen, place a mark on the glass plate 1 cm below
the lower edge of the comb to highlight the level to which the
resolving gel is poured. Remove the comb (see Fig. 1a).
3. Prepare the required volumes of 7.5% resolving gel (see Notes
13 and 14) and stacking gel in a small beaker or a capped tube
(see Table 1), without adding APS and TEMED (see Note 15).
Add 10% APS and TEMED into the resolving gel, swirl gently
to mix and pipette (or gently pour) the resolving gel into the
gel cassette up to the marked line. Immediately, add a few
drops of distilled water gently over the resolving gel using a
pipette to make a thin layer (see Note 16) and leave aside with-
out disturbing for approximately 30 min to set.
4. Once the resolving gel is set (see Note 17), add 10% APS and
TEMED to the stacking gel, gently swirl (or invert if it is a
tube) to mix, discard the distilled water layer, and pipette the
stacking gel over the resolving gel. Gently insert the comb
(thickness 1.0 mm 10 well) immediately after adding the stack-
ing gel. Care should be taken not to trap air under the comb
while inserting the comb. Allow approximately 30 min with-
out disturbing to set (see Note 17).
5. Once the gel is set (see Note 17), mix 20 μL sample (see Note
18) with 10 μL sample buffer just before the sample loading
(see Notes 14 and 19).
6. Load the prepared samples into the wells (see Note 20)
using gel loading tips. Electrophoresis is carried out at
Fig. 1 Polyacrylamide gel electrophoresis. (a) Preparation of the gel without SDS and the substrate, (b) loading
the samples without SDS and reducing agents, (c) electrophoresis at 4 °C
4 °C under constant current of 20 mA until the dye front

reaches the lower end of the gel or as desired (see Note 21)
(see Fig. 1b and c).
3.3 Detection 1. After electrophoretic separation, enzyme activity was detected,

of Enzyme Activity based on previously published methods [8, 13, 14]. Once the
Using Zymography electrophoresis is finished, disconnect the power supply. Invert
the glass plate with the gel, gently loosen the gel from the glass
plate from one edge using a spatula, and float it in a small con-
tainer (see Notes 22 and 23) with the formate buffer. Gently
swirl and discard the buffer, add formate buffer again, and
leave the gel to equilibrate with the buffer for 10 min (see Note
24). Discard the formate buffer.
2. Incubate the gel with 1% bovine hemoglobin at pH 3.0 for
15 min. Subsequently, wash the gel once gently with the for-
mate buffer (pH 3.0) and incubate for 1 h at 37 °C in a humid
container (see Notes 25 and 26) (see Fig. 2a and b).
Table 1
Composition of the gel components
Stacking
Components Resolving gel (7.5%)a gel (4%)
Water 3.7 mL 3.05 mL
1.5 M Tris–HCl (pH 8.8) 1.875 mL –
0.5 M Tris–HCl (pH 6.8) – 1.25 mL
Acrylamide (30%), bisacrylamide 1.875 mL 660 μL
(0.8%)
Add the following reagents immediately before dispensing into the gel
cassette
10% ammonium persulfate (APS) 37.5 μL 50 μLb
Tetramethyl ethylenediamine 3.75 μL 5 μLb
(TEMED)
Volumes given are sufficient to prepare 1 gel
a
b
APS and TEMED are added to the stacking gel mixture only when the resolving mini
gel is set
Fig. 2 Zymography. (a) Addition of the substrate (bovine hemoglobin at pH 3.0), (b) incubation of the gel with
the bound substrate in a humid chamber for hydrolysis to occur, (c) gel is stained with amido black, (d) Aspartic
proteinase activities are detected after destaining the gel
Fig. 3 Detection of multiple aspartic proteinase activities from a tissue extract.

Arrows show the multiple aspartic proteinase activities (see Ref. 9)
3. Add the amido black stain to the gel container and keep on a
shaker for 30 min. Remove the stain (see Note 27) (see Fig. 2c),
add destain, and leave overnight (see Note 28).
4. Keep the gel container on a light box and visualize the clear
areas in a dark background (see Note 28) (see Figs. 2d and 3).
4 Notes
1. As the pH of the Tris buffer changes significantly with the tem-

perature, pH should be adjusted after storing the buffer at
4 °C as the electrophoresis is conducted at 4 °C. It is better to
avoid concentrated HCl during the final adjustments of pH.
2. Caution: Acrylamide and bisacrylamide monomers are neuro-
toxic! Wear a mask and gloves and use a fume hood during
preparation. Prevent any spills during weighing. Acrylamide,
bisacrylamide reagent, is also available commercially.
3. APS stored at 4 °C can be used for a few times within a couple
of weeks.
4. Do not adjust the pH of the ×10 and ×1 running buffer.
Prepare the ×1 running buffer fresh and store at 4 °C. SDS is
not added to the running buffer.
5. Prepare the hemoglobin solution fresh immediately after start-
ing the electrophoresis and keep it stirring using a magnetic
stir bar to dissolve. This may take approximately 1 h. The sub-
strate was brought to the optimum pH (pH 3.0) of the aspar-
tic protease.
6. It is extremely important to take precautions to preserve the
enzyme activity until the end of the reaction with the substrate
in zymography. Avoid heat, detergents, foaming, and over-

dilution to minimize loss of activity. All procedures involving
the samples such as collection, transport, extraction, and elec-
trophoresis should be conducted at 4 °C.
7. Avoid bringing the sample near the optimum pH of the pro-
teinase during extraction and storage, to avoid activation of the
proteinase which might cause auto-degradation of the prote-
ase. A buffer at pH 7.4 would be fine for aspartic proteases.
8. Protease inhibitors should not be added when cell lysates are
prepared.
9. Tissue extracts should be preferably stored at −80 °C until the
preparation of the polyacrylamide gel is complete. Avoid
repeated thawing of the frozen samples.
10. It is necessary to assess the optimum pH of the aspartic pro-
teinase and the activity prior to starting the procedure as this
information is necessary to select the pH and the incubation
period for the zymography.
11. Since the protein migration during PAGE depends on the net
charge, it is important to know that the protein under investi-
gation is negatively charged during the separation (most of the
proteins are negatively charged at pH 8.5). In case that the pI
of the protein is above 8.5, the two electrodes should be
reversed when connecting to the power supply to prevent a
backward movement. However, reported pIs of aspartic pro-
teinases are less than 8.5. Aspartic proteinases identified from
the porcine ovarian extracts showed a movement towards the
anode indicating that their pIs are less than 8.5.
12. After assembling the gel cassette, fill the cassette with distilled
water using a wash bottle and leave for about 5 min to ensure
there is no leaking. Discard distilled water before pipetting the
gel solution into the cassette.
13. Some proteases may give better results with 10% resolving gels.
To get the desired percentage, adjust only the volumes of
acrylamide, bisacrylamide solution, and distilled water (see
Table 1).
Gel percentage = (Volume of acrylamide, bisacrylamide
solution/Total volume) × 30.
14. Sodium dodecyl sulfate and β-mercaptoethanol should be
omitted from all reagents.
15. TEMED and APS are the polymerizing agents and therefore
are added only when the resolving and stacking gels are about
to pour into the cassette. As the stacking gel is added only
when the resolving gel is set, delay the addition of TEMED
and APS until such time.
16. Addition of a thin layer of distilled water will leave a smooth

and even edge on the resolving gel and prevent drying during
the period of setting.
17. Approximately 5.6 mL resolving gel is poured into the gel cas-
sette with a 1 mm spacer for each mini gel. Resolving and
stacking gels were prepared with extra volume (7.5 mL resolv-
ing gel) to retain extra gel in the container after pouring into
the gel cassette to monitor whether the gel is set. Do not han-
dle the gel cassette to check for gel setting as it might cause
formation of uneven gels.
18. Total protein concentration of the samples can be determined
by the standard Bradford method [15] with bovine serum
albumin as the standard.
19. Do not boil the sample after adding sample buffer. SDS is not
added to the sample buffer.
20. It is better to load the samples well apart to leave sufficient
space to view the catalytic activity (see Fig. 1b). The capacity of
a well is 30 μL, if a 10 well comb with a 1 mm thickness is used.
Better is not to use more than 90% of the maximum volume of
a well to prevent overflow of the sample to adjacent wells. Add
sample buffer alone onto the empty wells.
21. If the proteases have a lesser mobility, the duration of electro-
phoresis can be prolonged after the dye front leaves the gel.
22. Handle the 7.5% gel with care as the gel is fragile. Before
removing the gel from the glass plate, mark one end of the gel
to identify the lanes and separate the stacking gel. As a practice,
I make a small cut at the lower end of the right side (towards
the last well) (see Fig. 2c and d) of the gel.
23. Use a transparent container with a lid which is little larger than
the gel. Use one container for each gel. A volume of 20 mL of
buffer, substrate, stain, and destain is sufficient for each gel.
24. At the end of the electrophoresis, wash and equilibrate the gel
in formate buffer (pH 3.0) in order to bring the gel to the
acidic pH required to activate the aspartic proteases.
25. Keep two or three damp cotton wool plugs (or paper towels)
along the sides of the gel container to prevent the gel from
drying during the incubation (see Fig. 2b). Keep the container
closed during the incubation. An incubator or a water bath can
be used (take precautions to prevent water leaking into the
container if a water bath is used).
26. The duration of incubation in the humid container after
removing the substrate can be adjusted to suit the desired
results. Incubation period will depend on the activity of the
proteases. Even though a longer incubation increases the sen-
sitivity, it might result in broader bands due to diffusion of
proteins, decreasing the resolution. Broader bands might mask

multiple proteinase activities found in close proximity, while a
shorter incubation may not reveal sufficient visibility of clear
areas. Duration of proteolysis should be strictly controlled if
the data are to be used in a quantitative manner.
27. Amido black stain can be reused for several times.
28. Partial destaining will occur within a much shorter time with
visible results. I prefer to use the gel for viewing and imaging
after an overnight destaining. Clear areas observed with
zymography correspond with the aspartic proteinase bands
with different net charges (see Figs. 2d and 3). Make images for
the future reference.
References
1. Kandapur R (2013) Zymography: enzymes in 8. Barrett AJ (2013) In: Rawlings ND, Woessner
action. Sci Int 1(4):70–75 JF (eds) Handbook of proteolytic enzymes.
2. Vandooren J, Geurts N, Martens E, Van den Academic Press. Elsevier, London
Steen PE, Opdenakker G (2013) Zymography 9. Perera HKI, Fernando PHP, Athauda SBP
methods for visualizing hydrolytic enzymes. (2015) Zymographic detection of aspartic pro-
Nat Methods 10(3):211–220 teinase activities in porcine ovarian extracts. Int
3. Wilkesman J, Kurz L (2012) Advances in J Biochem Res Rev 7(4):166–174
zymography techniques and patents regarding 10. Ornstein L, Davis BJ (1964) Disc electropho-
protease analysis. Recent Pat Biotechnol resis-I: background and theory. Ann N Y Acad
6(2):106–114 Sci 121:321–349
4. d’Avila-Levy CM, Santos ALS, Cuervo 11. Laemmli UK (1970) Cleavage of structural
P, de Jesus JB, Branquinha MH (2012) proteins during the assembly of the head of
Applications of zymography (substrate- bacteriophage T4. Nature 227:680–685
SDS-PAGE) for peptidase screening in a 12. Ausubel FM, Brent R, Kingston RE, Moore
post-genomic era. In: Magdeldin S (ed) Gel DD, Seidman JG, Smith JA, Struhl K (eds)
electrophoresis—advanced techniques. ISBN: (2003) Current protocols in molecular biol-
978-953-51-0457-5. InTech. Available from ogy. John Wiley & Sons, New York
http://www.intechopen.com/books/gel- 13. Takahashi K, Matsumoto K, Nishii W,
electrophoresis-advanced-techniques/application Muramatsu M, Kubota K, Hachioj HI et al
sofzymography-substrate-sds-page-for- (2009) Comparative studies on the acid pro-
peptidase-screening-in-a-post-genomic-era teinase activities in the digestive fluids of
5. Pan D, Hill AP, Kashou A, Wilson KA, Tan- nepenthes, cephalotus, dionaea, and drosera.
Wilson A (2011) Electrophoretic transfer protein Carniv Pl Newsl 38:75–82
zymography. Anal Biochem 411(2):277–283 14. Furihata C, Kawachi T, Sugimura T (1972)
6. Arndt C, Koristka S, Bartsch H, Bachmann M Premature induction of pepsinogen in develop-
(2012) Native polyacrylamide gels. Methods ing rat gastric mucosa by hormones. Biochem
Mol Biol 869:49–53 Biophys Res Commun 47:705–711
7. A guide to polyacrylamide gel electrophoresis 15. Bradford M (1976) A rapid and sensitive method
and detection. http://www.bio-rad.com/ for the quantification of microgram quantities of
webroot/web/pdf/lsr/literature/ protein utilizing the principle of protein-dye
Bulletin_6040.pdf. Accessed 10 Feb 2016 binding. Anal Biochem 72:248–254
Chapter 6
MMP Activity Detection in Zymograms

Péter Bencsik, Monika Bartekova, Anikó Görbe, Krisztina Kiss,
János Pálóczi, Jana Radosinska, Gergő Szűcs, and Péter Ferdinandy
Abstract
Matrix metalloproteinases (MMP) belong to a distinguished class of zinc-dependent endopeptidases.
Zymography is a semi-quantitative tool for determining the activity of different MMP isoenzymes in a
variety of biological samples. In substrate gel zymography, protein samples of different origin (tissue, cell
lysates, plasma/serum, perfusates, other liquids) are separated in sodium dodecyl sulfate (SDS) polyacryl-
amide gels containing copolymerized substrate (gelatin, casein, elastin, etc.), and after incubation-enabling
substrate cleavage by MMPs, MMP activities are detected after the gel staining as transparent bands against
a dark-blue background. In situ zymography is a histological modification of substrate zymography in
frozen sections, allowing detection of the localization of the MMP activities within the tissue. Here, we
describe detailed experimental protocols of all abovementioned techniques and provide examples for
several sample measurements.
Key words Matrix metalloproteinase activity, Substrate zymography, Gelatin, Casein, In situ zymography
1 Introduction
1.1 Matrix Matrix metalloproteinases (MMPs) are enzymes that belong to the
Metalloproteinases family of zinc-dependent endopeptidases and are known to play a
in Health and Disease crucial role in the dynamic processing of the extracellular matrix
(ECM) facilitating the degradation of matrix material. On the other
hand, MMPs have been also shown to be present intracellularly,
thereby influencing physiological as well as pathological intracellular
signal transduction processes and the contractile machinery.
Therefore, MMPs are interesting drug targets for several pathologies
(see for a review: [1]). The family of MMPs include close to 30 mem-
bers (see for reviews: [2, 3]); many of them shown to be activated due
to different physiological as well as pathological situations in different
tissues. Some of the important family members together with their
characteristic features are shown in Table 1. MMPs are synthesized as
zymogens and can be activated by proteolytic cleavage of an amino-
terminal domain, by oxidative/nitrosative-induced conformational
53
54 Péter Bencsik et al.
Table 1
The family of MMPs and their characteristic features
Molecular weight
(kDa)
MMP
codes Alternative names Za Aa Substrates Pathologies
MMP-1 Interstitial 57 52 Gelatin Atherosclerosis,
collagenase melanoma, heart
failure
MMP-2 Gelatinase A, 75/72 64 Gelatin, elastin Myocardial infarction,
type IV collagenase heart failure, gastritis,
rheumatoid arthritis
MMP-3 Stromelysin-1 57 45 Gelatin, elastin, Brain injury,
casein neurodegeneration
MMP-7 Matrilysin 28 19 Gelatin, elastin, Tumor-induced
casein osteolysis, colon
cancer
MMP-8 Neutrophil 75 57 Gelatin Coronary artery disease,
collagenase angina
MMP-9 Gelatinase B 92 86 Gelatin, elastin Myocarditis and
subsequent dilated
cardiomyopathy,
ulcerative colitis
MMP-10 Stromelysin-2 57 44 Gelatin, elastin, Lung cancer
casein
MMP-11 Stromelysin-3 51 44 Gelatin, elastin, Tumor progression,
casein breast carcinomas
MMP-12 Macrophage 54 22 Gelatin, elastin, Granulomatous skin
metalloelastase casein diseases,
inflammatory
disorders
MMP-13 Collagenase-3 65 48 Gelatin Breast carcinomas
MMP-14 MT1-MMP 66 54 Gelatin, casein Tumor growth by
activating MMP-2
MMP-15 MT2-MMP 76 N/A Fibronectin Obesity, preeclampsia,
Laminin ovarian carcinoma
MMP-16 MT3-MMP 64 52/30 Gelatin, casein Breast cancer
MMP-17 MT4-MMP 71 67 N/A Preeclampsia
MMP-19 RASI-1 57 N/A Gelatin Rheumatoid arthritis
MMP-20 Enamelysin 54 42.5 Amelogenesis
imperfecta
(continued)
MMP Activity Detection in Zymograms 55
Table 1
(continued)
Molecular weight
(kDa)
MMP
codes Alternative names Za Aa Substrates Pathologies
MMP-21 N/A N/A N/A N/A Melanoma, ovarian and
colon carcinomas
MMP-22 N/A N/A 42 N/A N/A
MMP-23 N/A N/A N/A N/A Breast cancer
MMP-24 MT5-MMP N/A N/A N/A Brain tumors
MMP-25 MT6-MMP, 56 38 pro-MMP-2 Inflammatory
leucolysin hyperalgesia
MMP-26 Endometase 29 19 Gelatin Lung cancer
Z and A indicates the zymogen or active form(s) of MMPs, respectively
a
change (without a change in molecular weight), or by phosphoryla-

tion [2, 4]. Activities of MMPs are tightly regulated by their endog-
enous tissue inhibitors (TIMPs) [5].
MMPs play an important role in many physiological and patho-
logical processes, including embryogenesis, wound healing, inflam-
mation, cardiovascular diseases, and tumor development or
progression [2, 6]. Increased activities of different MMPs have been
reported to be connected with different pathological situations such
as ischemia-reperfusion injury [7], myocardial contractile dysfunc-
tion [8], heart failure [9], arthritis [10], neurodegenerative disor-
ders [11], cancer invasion and metastasis [12], liver cirrhosis [13],
fibrotic lung disease [14], periodontal disease [15] as well as with
responses to some invasive interventions like anthracycline treat-
ment [16, 17] or chest irradiation [18] used in the cancer therapy.
On the other hand, inhibition of MMP activities has been shown to
be connected with some kinds of tissue protection such as ischemic
preconditioning [19, 20] or flavonoid- induced cardioprotection
[17]. Moreover, pharmacological inhibition of MMP activities has
been shown to be cardioprotective in animal models of acute myo-
cardial infarction [21, 22] and has also been shown to be altered in
coronary artery disease patients [23]. Being able to detect MMPs at
early stages of the disease is opening a perspective to use MMPs as
diagnostic markers. MMPs have been well-investigated in clinical
studies of cardiovascular diseases: MMP-2 and -9 in Chagas cardio-
myopathy [24] and MMP-9 in ST-segment elevation myocardial
infarction [25]. Protein expression and activation of MMP-2 and
MMP-9 has clinical relevance and prognostic value in patients with
colorectal cancer [26]. Fecal MMP-9 is a useful tool for the differen-
tial diagnosis of diarrheic disorders and in the noninvasive evaluation
of disease activity and mucosal healing in ulcerative colitis [27].
Regarding the abovementioned facts, determination of MMP

activities belongs to very useful methodologies in biomedical
research and is of high clinical importance since it seems to be a
powerful diagnostic and/or therapeutic tool for the detection or
follow-up of the abovementioned pathologies.
1.2 MMP Activity MMP activities can be determined by zymography in different

Detection in Biological kinds of biological samples such as heart, brain, liver, lung tissues,
Samples blood vessels, or in isolated or cultured cell lineages (see Figs. 1, 2,
and 3). Zymography can be performed as a substrate zymography,
in which the substrate of the certain MMP is incorporated, copo-
lymerized in a sodium dodecyl sulfate (SDS) polyacrylamide gel,
and MMPs are separated according to their molecular weights.
Fig. 1 Representative gelatin zymograms performed from human samples.

(Panel A) Human serum samples from patients with coronary artery disease.
Some of the patients have increased MMP-2 and/or MMP-9 activity as indicated
by the zymographic intensity at 72 or 86 kDa, respectively. (Panel B) Gelatin
zymogram from human isolated lymphocytes. 72 kDa MMP-2 activity is mark-
edly visible; however, other activities at 64 kDa (MMP-2) and at 86 kDa (MMP-9)
can also be observed. Weak gelatinolytic activity signals can be seen in both
panels at 192 kDa, which may indicate the dimerized form of MMP-9 [28]
Fig. 2 Representative gelatin zymograms performed from homogenates of

murine tissues (L1–4: lanes 1–4; 15 μg protein was loaded into each wells).
Panel A represents mouse heart homogenates, which expresses mainly the
72 kDa isoform of MMP-2. Weak signal for MMP-9 can be observed; however, it
indicates inappropriate removal of blood from heart samples. (Panel B) Lung
samples from mice subjected to chronic tobacco smoking (L2 and 4) and their
controls (L1 and 3). A markedly increased intensity can be observed at 92 kDa
(MMP-9). (Panel C) Samples derived from the aorta of transgenic mice. L1 and 2:
ApoB100LDL−/− mice, control and Chlamydia pneumoniae (Cpn) infected; L3
and 4: ApoE−/−, control and Cpn infected
The activity of MMPs is detected by the absence of gelatin in the

gel, which can be visualized by transilluminating the gel. The activ-
ity of the certain MMP is proportional with the intensity and the
thickness of the corresponding band on zymogram, which can be
evaluated electronically by using different software after scanning
the gels.
Fig. 3 Examples of MMP zymography in isolated and/or cultured cells. (Panel A) Gelatin zymogram from cultured
neonatal cardiac myocytes after resuspension. Gelatinolytic activities can be detected at completely different
molecular weights than that of heart homogenates (for comparison see Fig. 2a). (Panel B, C) In situ gelatin
zymography in fixed neonatal cardiac myocyte culture in normoxic conditions (Panel B) and subjected to simu-
lated ischemia/reoxygeation (Panel C). Panel C shows an increased gelatinolytic activity (represented as green
fluorescence—DQ™ fluorescent gelatin; red fluorescence: MMP-2 immunostaining by rhodamine-labeled goat
anti-mouse antibody, blue fluorescence: cell nuclei by Hoechst 33342 staining) in cardiac myocytes, which
indicates the presence and activation of MMP-2 during simulated ischemic stress. Scale bars = 20 μm
For the detection of MMP activity in situ in different cell or

tissue cultures, in situ zymography is a suitable method (Fig. 3). In
in vivo systems, natural inhibitors of MMPs (tissue inhibitors of
MMPs, TIMPs) are presented [29]. Therefore, when a study is
designed for detecting MMPs activity in a certain physiological or
pathological condition, one should calculate not only with the acti-
vation, but the inhibition of MMPs by TIMPs as well. For this
reason, reverse zymography has been developed, which allows
detection of TIMPs activities in gel zymograms. However, this
chapter is limited for showing the opportunities to detect MMP
activities in biological samples, thus here we do not provide detailed
description on the available techniques for detecting TIMPs activ-
ity (for TIMP measurements, see for review [30]).
1.3 Types MMPs cleave different substrates; therefore, the substrate, which is
of Zymography copolymerized in the gel, should be determined according to the
Substrates MMP isoform.
Gelatin zymography is predominantly used for measurements
of activities of MMP-2 and MMP-9 as these two MMPs exert
strong ability to cleave gelatin as a substrate and are commonly
called “gelatinases.”
In casein zymography, casein is copolymerized into the poly-
acrylamide gel as a substrate for MMP cleavage. Casein zymogra-
phy is used for estimation of proteolytic activity of MMP-7 due to
its ability to cleave casein.
The most rarely applied type of zymography is elastin zymog-
raphy, in which soluble elastin is copolymerized in the gel and,
beside the activity of elastases, it can suitably show elastinolytic
activity for such MMPs, which cleave basically other substrates like
gelatin (e.g., MMP-2).
2 Materials
2.1 Sample 1. For pulverized tissue samples: Homogenization buffer: 500 mL

Preparation double distilled water (ddH2O), 0.335 g (50 mM) Tris base,
1 mL (0.5%) Triton X-100. In 500 mL beaker dissolve com-
pounds in 500 mL ddH2O. Adjust to pH 7.4 with 1 M HCl.
Aliquot into 15 mL Falcon tubes. Store at −20 °C for 1 year.
Homogenator: Pellet Pestle Motor, Centrifuge (e.g., Hettich
Universal 320R), BCA Protein Assay Kit for protein
measurement.
2. For organ perfusates: Concentrating tubes: Amicon Ultra-4
30 kDa centrifugal filter unit with Ultracel-30 membrane for
perfusate concentration.
3. For cell culture lysates: Phosphate Buffered Saline tablets (PBS):
In 200 mL beaker dissolve 1 PBS tablet in 100 mL ddH2O. Adjust
to pH 7.2 with 1 mM NaOH prepared freshly; Homogenization

buffer (see above item 1); Amicon Ultra 10 kDa concentrating
tubes. Centrifuge (e.g., Hettich Universal 320R)
2.2 Gelatin 1. 30% Acrylamide/0.8% Bisacrylamide, Store: at +4 °C for

Zymography 1 year.
2. Separating Gel Solution—1.5 M Tris–HCl, pH 8.8, Store at
+4 °C for 1 year.
3. 2% Gelatin Solution: 100 mg gelatin (type A, from porcine
skin; stored between 20–25 °C) dissolved in 4.5 mL ddH2O.
4. 10% (w/v) SDS (sodium dodecyl sulfate) solution. Weigh 10 g
SDS and dissolve in 100 mL ddH2O (Storage between
20–25 °C for 1 year).
5. 10% (w/v) ammonium persulfate (APS) solution: Dissolve
100 mg of APS in 1 mL ddH2O. Storage: at +4 °C for 1 month
6. TEMED. Storage: at +4 °C for 1 year.
7. Stacking Gel Solution: 0.5 M Tris–HCl/SDS, pH 6.8. Storage:
at +4 °C for 1 year.
8. ELFO Buffer (25 mM Tris–HCl, 192 mM glycine, 0.1% SDS,
pH 8.3). Storage: at +4 °C for 1 year. Or self-prepared: 28.83 g
glycine, 6.0 g Tris base, 2.0 g SDS. Dissolve Tris base and gly-
cine in 1000 mL of ddH2O. Bring solution to 1950 mL with
ddH2O. Add SDS. Bring solution to 2000 mL total volume
with ddH2O. Storage: at 4 °C for 1 year.
9. Non-reducing loading (sample) buffers: use commercial
“Blue” Zymogram Sample Buffer or commercial “Pink” non-
reducing lane marker.
10. For positive control, use “zymography standard,” containing a
mixture of purified and activated MMP-2 and MMP-9; or
MMP-2 standard for gelatin and elastin zymography (Fig. 1a).
For casein zymography, human, recombinant active MMP-7
can be used.
11. Renaturation buffer: Renaturation buffer (Bio-Rad). Store: at
+4 °C for 1 year. Add 1× mL Renaturation buffer (Bio-
Rad) + 9× mL ddH2O. Storage: prepare freshly.
12. Development buffer: Development buffer (Bio-Rad) Storage:
at +4 °C for 1 year. Add 1× mL Development buffer + 9× mL
ddH2O. Storage: prepare freshly.
13. For negative control, use 10 mM ethylene glycol tetraacetic
acid (EGTA, binds Ca2+ ions, which is obligatory for activity of
MMPs). Dissolve 381 mg EGTA in 90 mL ddH2O + 10 mL
development buffer.
14. Coomassie Brilliant Blue (0.05%): 250 mg Coomassie Brilliant
Blue G-250, 125 mL methanol, 50 mL glacial acetic acid,
325 mL ddH2O. Dissolve 250 mg Coomassie Brilliant Blue in

the mixture of 125 mL methanol and 50 mL glacial acetic acid
and dilute it by adding 325 mL ddH2O. Storage: between
20–25 °C.
15. Destaining solution: 40 mL methanol, 80 mL acetic acid,
880 mL ddH2O. Storage: between 20–25 °C
2.3 Casein All materials and procedures are identical with gelatin zymography
Zymography except gelatin solution, which is substituted with casein.
1. Casein solution: 100 mg casein (storage: between 20–25 °C)
dissolved in 4.5 mL phosphate buffer.
2. Phosphate buffer (126 mM).
2.4 Elastin All materials and procedures are identical with gelatin zymography
Zymography except gelatin solution, which is substituted with elastin.
1. Elastin solution: 54 mg soluble elastin from bovine neck liga-
ment dissolved in 4.5 mL ddH2O.
2.5 Preparation 1. Gelatin solution: Add 100 mg gelatin to 4.5 mL ddH2O. Gently

of Substrate heat and mix solution until it dissolves (beaker will be warm to
for Electrophoresis touch, max. 40 °C). If it has cooled down, add 0.5 mL 10%
(w/v) SDS aqueous solution to reach final desired volume and
concentration. Prepare freshly (see Note 1).
2. Casein solution: Dissolve 100 mg casein in 4.5 mL phosphate
buffer. Stir until casein dissolves. Preparation of phosphate
buffer: mix 7.12 g Na2HPO4 dissolved in 400 mL ddH2O and
1.56 g NaH2PO4 dissolved in 100 mL ddH2O, set at pH 7.4.
Add 0.5 mL 10% w/v SDS aqueous solution to reach final
desired volume. Alternatively, dissolve 30 mg casein in 2 mL of
75 mM Tris–HCl, pH 8.8.
3. Elastin solution: Add 54 mg κ-elastin to 4.5 mL ddH2O. Stir
until elastin dissolves. Add 0.5 mL 10% w/v SDS aqueous
solution to reach final desired volume.
2.6 In Situ The EnzCheck Gelatinase/Collagenase Assay Kit from Invitrogen

Zymography was used. This assay includes the following reagents:
1. DQ gelatine from pig skin: five vials (1 mg DQ substrate
lyophilized from 1 mL of PBS in each vial).
2. 10× Reaction buffer: 50 mL.
3. 1,10-phenanthroline monohydrate: 30 mg powder in a vial.
4. Collagenase type IV from Clostridium histolyticum: 500 U col-
lagenase powder in a vial.
3 Methods
3.1 Sample 1. Weigh out 30–50 mg heart (lung, pancreas, aorta, spleen) tis-
Preparation sue powder into a liquid-nitrogen-frozen 1.5 mL Eppendorf
tube. Avoid thawing. It can be stored at −80 °C for 2 years.
3.1.1 Pulverized
Tissue Sample 2. Thaw an appropriate amount of homogenization buffer
(see Note 2).
3. Add 4× volume homogenization buffer to the sample (e.g.,
30 mg sample and 120 μL buffer).
4. Homogenize the mixture by Pellet Pestle Motor for 3 × 10 s
(see Note 3).
5. Centrifuge the homogenate at 4 °C for 10 min at 10,000 × g,
and collect the supernatant, and store at −80 °C for maximum
1 month.
6. Measure protein concentration by a BCA kit. Usually 20× dilu-
tion of tissue homogenates is required.
3.1.2 Preparing 1. In case of perfusate sample, use Amicon Ultra 30 kDa concen-
Perfusate Samples trating tubes to concentrate 4 × 3 mL perfusate sample to
50–100 μL.
2. Pour 3 mL perfusate in the concentrating insert of an Amicon
tube.
3. Put concentrating insert into the tube and close it. Spin sam-
ples at 7500 × g for 20 min, 4 °C.
4. Remove the concentrating insert and discard the flow-through
from the tube. Reinsert the concentrating insert.
5. Pour 3 mL perfusate in the insert, recap, and spin it for 20 min.
Repeat steps 3–4 twice more.
6. Pipette out the concentrated sample from the insert into an
Eppendorf tube.
7. Measure protein concentration by BCA kit. Usually 3× dilu-
tion of perfusate concentrates is required.
3.1.3 Cell Culture 1. For cell culturing see Ref. 31 (culturing neonatal cardiac
Lysates myocytes).
2. Remove treating solutions and wash cells in 2 mL PBS two
times and then remove PBS.
3. Scrape cells from two wells of a 6-well plate in 200 μL zymog-
raphy homogenization buffer (two wells together are 400 μL
and they are collected into one tube after washing both wells).
In case of 25 cm2 flask use 400 μL buffer; in case of 75 cm2
flask, use 1 mL buffer.
4. Keep Eppendorf tubes with the suspensions on ice and take
into −80 °C freezer or concentrate them freshly.
3.2 Concentration 1. Keep samples on ice.

of Samples 2. Homogenize the mixture by ultrasonic homogenizer 2× 5 s on
ice.
3. Centrifuge cell homogenates at 5000 × g for 10 min at 4 °C.
4. Collect supernatant.
5. Centrifuge the supernatant in Amicon Ultra 10 kDa concen-
trating tubes to increase the sample protein concentration
(4000 × g for 30–50 min at −4 °C).
6. Put 50–100 μL samples into −80 °C freezer in two aliquots
(one aliquot for determination of protein concentration).
7. Measure protein concentration by BCA kit. Usually 3× dilu-
tion of cell concentrates is required.
3.3 Preparation 1. Assemble electrophoresis unit (see Note 4).

of Separating Gel 2. Insert a comb between the gel-casting glasses and mark desired
level of separating gel on the front glass (at the bottom of the
teeth of the comb).
3. Mix 30% acrylamide/0.8% bisacrylamide solution with Tris–
HCl pH 8.8, gelatin solution, and ddH2O (Table 2; see Notes
5 and 6).
4. Add 10% APS solution and TEMED to the mix quickly
(Table 2).
Table 2
Preparation of separating gel between 0.75–1.5 mm thickness
Final acrylamide concentration in the separating gel (%)
8.0
Small gel Large gel

Stock solutionsa 7.0 7.5 (15 mL) (22.5 mL) 9.0 10.0
30% acrylamide/0.8% 3.5 3.75 4.0 6 4.5 5.0
bisacrylamide (mL)
1.5 M Tris–HCl, pH 8.8 (mL) 3.75 3.75 3.75 5.62 3.75 3.75
ddH2O (mL) 6.25 6.0 5.75 8.62 5.25 4.75
Gelatin/casein/elastin solution 1.5 1.5 1.5 2.25 1.5 1.5
(20/20/12 mg/mL, 1% w/v
SDS; mL)
10% w/v Ammonium Persulfate 50 50 10 75 50 50
Solution (APS; μL)
TEMED (μL) 10 10 10 15 10 10
See Notes 8 and 9
a
5. Swirl to get homogenous gel. Avoid bubbling. Use immedi-

ately as polymerization process has begun (see Note 7).
6. Using a pipette, pour a small amount into sandwich plates and
watch for leakage. In the absence of leakage, continue filling
up to 1 mm above line.
7. Gently add butanol (with a 27G-needle connected to a
10-mL syringe) along the surface of the gel to remove bub-
bles (see Note 8).
8. Allow gel to polymerize (approximately 20 min at 25 °C; see
Note 9). Use this time to prepare stacking gel (without adding
TEMED and 10% APS).
9. A layer of H2O on the surface of the gel will be visible when
polymerization is completed. Drain this layer from the unit
with a small stripe of blotting/filter paper.
3.4 Preparation 1. Mix 30% acrylamide/0.8% bisacrylamide solution with Tris–

of Stacking Gel HCl, pH 6.8 and ddH2O (Table 3).
2. Add 10% SDS, 10% APS, and TEMED quickly (Table 3).
3. Swirl to mix. Avoid bubbling. Use immediately as polymeriza-
tion process has begun.
4. Place comb in units and then use pipettes to pour stacking gel.
5. Allow gels to polymerize (approximately 15 min at 25 °C).
Use this time to make sample calculation.
3.5 Sample 1. According to the results of protein measurement, sample load-

Calculation ing mass and volume should be calculated.
2. From a tissue homogenate, 50 μg protein per lane should be
loaded. Since we load 15 μL per lane, this means that the final
protein concentration of sample needs to be 50 μg/15 μL.
3. In case you want to load a sample only once, it is enough to
prepare 2× volume of one load (30 μL), which means that we
Table 3
Preparation of 5 mL stacking gel
Stock solutions Volume

30% acrylamide/0.8% bisacrylamide 1 mL
0.5 M Tris–HCl pH 6.8 1.25 mL
ddH2O 3.05 mL
10% SDS 50 μL
10% w/v Ammonium Persulfate Solution (APS) 25 μL
TEMED 8 μL
should add 6 μL (1/5 part) “pink” or 20 μL (2/3 part) “blue”
loading buffer.
4. The remaining 24 or 10 μL, respectively, should contain
2 × 50 μg = 100 μg protein. Therefore, volume of the sample
will be: V1 = 100 μg/protein concentration of your sample.
5. Then we should add ddH2O to dilute samples. The required
volume of ddH2O is: V2 = 30 − V1.
Taken together:
Loading volume: 15 μL
Loaded protein: 50 μg
Prepared volume: 30 μL (2× loading volume)
Prepared (sample) 2× loaded protein (100 μg)
mass:
“Pink” loading 1/5 of prepared volume (6 μL) OR
buffer:
“Blue” loading 2/3 of prepared volume (20 μL)
buffer:
Sample volume: V1 = 2× loaded protein (2× 50 μg)/
sample protein concentration (μg/μL)
ddH2O: V2 = Prepared volume (30 μL) − V1
3.6 Sample Loading 1. Prepare and cool down ELFO buffer: 50 mL ELFO + 450 mL
and Running Gels ddH2O (avoid bubbles, mix it gently) (see Note 10).
2. Mark gel lanes for sample loading. Do not use the two outside
lanes.
3. When gel is polymerized, remove combs by pulling it straight
up.
4. Remove gel plates and snap onto electrode assembly
(see Note 11).
5. Fill up the lower and the upper buffer container with tank
buffer.
6. In case of casein zymography: pre-run electrophoresis at
40 mV for 15 min at 4 °C before the samples are loaded into
the wells. Another possibility is to load sample buffer into one
well and pre-run electrophoresis at 4 °C until it reaches the
bottom of the gel. Afterwards continue with step 7, but keep
gels at 4 °C (see Note 12).
7. Load samples
For identification of different isoforms of detected MMPs,
positive controls (e.g., zymography standard containing
human MMP-2 and -9 or MMP-2 standard) should be used.

Page ruler is also useful to detect the different size of bands.
Leave at least one lane loaded with sample(s) for negative con-
trol (see in Subheading 3.6).
8. Connect electrodes properly (red to red, black to black) and
set to 90 V.
9. Let the samples run until loading buffer (running front)
reaches the bottom of the gel (see Note 13).
10. Use this time to prepare renaturation and development
buffers.
3.7 Washing 1. Set dry incubator at 37 °C.

and Incubating Gels 2. Disassemble the casting apparatus.
3. Cut down lanes for negative control (see Note 14).
4. Wash gels for 40 min in 200 mL renaturation buffer between
20–25 °C (see Notes 15 and 16).
5. Place gels into 200 mL freshly prepared development buffer (see
Note 16). For negative control, incubate lane(s) separately in
development buffer substituted with 10 mM EGTA solution.
6. Incubate gels in dry incubator at 37 °C for 20–40 h (see Note 17).
3.8 Staining Gels 1. Immerse gels into 0.05–0.1% Coomassie Brilliant Blue solu-
and Preparation tion. Place on a shaker for 1 h (see Note 18).
for Evaluation of MMP 2. Incubate gels in destaining solution and place on a shaker (at
Activity least for 60 min; see Notes 19 and 20).
3. Gelatinolytic activities should be detected as transparent bands
against the blue background of Coomassie Brilliant Blue-
stained gelatin (see Figs. 1–3).
4. Scan the gel in transilluminator mode with a special gel scanner
or gel-documentation system.
5. Evaluate MMPs activity by using a gel/film evaluation soft-
ware (e.g., Quantity one, Bio-Rad).
3.9 In Situ The present description provides details for performing in situ
Zymography zymography for MMP-2 in cardiac myocytes (Fig. 3); however,
techniques for showing other MMP activities in different tissue
sections or cell cultures in situ also exist and a description for gen-
eral in situ zymography is available in Ref. 32.
1. Culture neonatal rat cardiac myocytes [31] or other cell types
in 24-well tissue culture plate at the density of 105 cells/well
for 3 days.
2. Replace the growth medium (DMEM—Dulbecco’s Modified
Eagle Medium—supplemented with Glu, AB/AM, 1% FBS)
with a “stress” solution containing DQ substrate at 40 μg/mL

concentration. In case of control group, replace the medium of
the cells with a control solution containing DQ substrate at the
abovementioned concentration.
3. Subject cells to circumstances according to your aim/project,
which may induce intracellular MMP-2 activation.
4. For negative control samples, use ilomastat (at 0.5 μM final
concentration), or other non-specific MMP inhibitor (e.g.,
1,10-phenanthroline, between 1 and 5 mM final concentra-
tion, SB-3CT between 1 and 10 nM final concentration).
5. Subsequently, replace “stress” solution with growth medium
containing DQ substrate at 40 μg/mL concentration (250 μL
volume must be applied onto the cells).
6.
Replace the medium, and wash cells twice with
Dulbecco’s-PBS.
7. Rinse cells in 3.7% paraformaldehyde in PBS between 20–25 °C
for 15 min.
8. Wash cells twice with PBS.
9. Rinse coverslips with mounting medium and view fluorescent
signal under fluorescent microscope.
10. If it is necessary, you can combine in situ zymography with
immunocytochemistry. In this case, after the fixation you
should continue with an appropriate immunostaining
protocol.
4 Notes
1. Gelatin is very sensible. Make sure that gelatin dissolves com-

pletely (clear, transparent solution without any opalescent par-
ticle). Avoid gelatinization: prolonged cooling leads to gel
formation. When SDS is added, avoid precipitation. If gelatin
precipitates, try to heat again. If precipitates do not disappear
from the solution, prepare a new gelatin solution. Precipitated
gelatin does not polymerize homogenously in the gel, which
may lead to equivocal results.
2. Avoid reducing agents (e.g., dithiothreitol; DTT) or protease
inhibitors (e.g., phenylmethanesulfonyl fluoride; PMSF) in the
homogenization buffer. They may reduce or inhibit MMP
activities, thereby may lead to false results.
3. Avoid ultrasonic homogenizer. It can destroy the native struc-
ture of MMPs; therefore, enzymes may lose their activities.
4. 7.5–10% polyacrylamide gels are recommended for gelatin
zymography. For casein zymography, we recommend to use
10–15% gels according to the MW of active MMP-7 (19–

21 kDa; 28–30 kDa pro-MMP-7).
5. Mix 15.0 mL of separating gel. This volume is sufficient for 1
small unit (containing ten lanes; i.e., two gels), or mix 22.5 mL
for a triple-wide unit (containing 30–34 lanes).
6. In order to avoid leakage, ensure that spacers, comb, and glass
plates are aligned properly.
7. Make sure that gel is horizontal and there is no difference in
the levels of the two edges.
8. Avoid “shooting” of butanol. Butanol should cover the whole
surface of the gel uniformly.
9. TIP: leave pipette tip in the remnant of separating gel; when it
is polymerized, you will be able to lift it with the pipette.
10. Calculate the necessary volume of ELFO buffer according to
the buffer tank.
11. Eliminate bubbles under the gel, they may disturb gel
running.
12. Casein migrates in gel during electrophoresis. Due to its low
molecular weight (23 kDa), the zone containing casein can
obscure MMP-7 (latent form: 29 kDa, active form: 20 kDa)
after staining. Therefore, pre-run of casein-embedded gel is
recommended before classical zymogram procedure in order
to get excess of casein out of the gel. The amount of remaining
casein is sufficient for detection of MMP activities [33].
13. Average time for gel running: 1.5–2 h.
14. Avoid gel rupture. Cut the different corner of the gels to be
able to identify the gels later (e.g., cut the bottom left corner
of gel #1, and both the top and bottom left corners for gel #2).
(Ensure that the gel is oriented correctly so that you don’t
accidentally cut the right side corner.)
15. Before preparation of renaturation and development buffers,
make sure that the buffers do not contain any visible contami-
nation (e.g., fungal particles). Buffers should be clear and
transparent.
16. Make sure that gels immerse in the buffers and are not attached
to the wall of the dish.
17. Even before staining, gelatinolytic activity can be visible. Hold
the gel up against a dark background to visualize it, if not,
longer incubation should be applied. A pilot zymography is
recommended to run for setting up the optimal incubation
time. For example, 20-h incubation is recommended in case of
human plasma, rat heart, and rodent lung samples, while 40-h
incubation is required for mouse heart samples.
18. Staining can be longer, if it is necessary. The gel should be dark

blue, protein ladder should be invisible.
19. Use clear dish and change destaining solution after 5–10 min,
if it becomes bluish.
20. Leave the gel in destaining solution until the stacking gel
becomes completely destained (transparent) again (see Figs. 1
and 3). It can last even for 12 h.
Acknowledgments
This work (P. Bencsik, A. Görbe) was supported by the János

Bolyai Research Scholarship of the Hungarian Academy of Sciences
and has received funding from the European Union’s Horizon
2020 research and innovation programme under grant agreement
No. 698297 Acronym: Infarnosys. P. Ferdinandy was a Szentágothai
Fellow of the National Excellence Program of Hungary
(TÁMOP-4.2.4.A/2-11/1-2012-0001).
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Chapter 7
Characterization of Novel Collagenolytic Proteases

Goran Mucić, Brankica Rašković, and Natalija Polović
Abstract
Collagenolytic proteases have many potential applications in different areas of science, industry, and medicine.
The determination of the activity of such proteins is paramount to their application. Here, we describe methods
which can be applied to determine the activity and some basic characteristics of potential collagenases.
Key words Collagenase, Collagen, Caseinolytic activity, Collagenolytic activity, BAPNA assay
1 Introduction
Proteases which cleave the triple helix of native collagen are called col-
lagenases. Mammalian collagenases can be divided into three sub-
groups, metallocollagenases (matrix metalloproteinases), serine
collagenases, and cysteine collagenases (e.g., human cathepsins), while
collagenases from different sources are found to belong to different
groups such as plant cysteine proteases, invertebrate serine and cyste-
ine proteases, and bacterial metallocollagenases. Metallocollagenases
contain zinc in their active site and sometimes require calcium for
stability and optimum activity [1, 2]. Many studies have been con-
ducted regarding metallocollagenases from mammalian and bacterial
sources. Serine collagenases are found in various marine species; they,
like mammalian and bacterial metalloproteases, possess collagenolytic
activity; however, they too possess a broad proteolytic activity [3].
Enzymes, such as collagenases, have many applications like
meat tenderizers, isolation and cultivation of mammalian cells, and
elimination of scar tissue; products of their enzymatic hydrolysis of
collagen can be used in lotions, as dietary supplements, etc.
Considering various applications of collagenases, it is natural that
there is a high interest in such proteins [4–20]. Detection and
characterization of these proteins is important not only for research
purposes, but for medical reasons as well. Matrix m etalloproteinases
are commonly detected via zymography in clinical practice since
these enzymes are involved in tumor invasion and metastasis [5].
71
72 Goran Mucić et al.
2 Materials
2.1 SDS-PAGE 1. Monomer solution: 30% acrylamide, 0.8% bisacrylamide.

and Gelatin SDS-PAGE Dissolve 58.4 g of acrylamide and 1.6 g of bisacrylamide in
100–120 mL of distilled water and, after dissolving, add dis-
tilled water to 200 mL. Filter the solution through filter paper
and store at 4 °C.
2. Running gel buffer: 1.5 M Tris–HCl buffer pH 8.8. Dissolve
36.3 g of Tris in 150 mL of distilled water. Use 4 M HCl to
bring pH value to 8.8 and add distilled water to 200 mL. Store
at 4 °C.
3. Stacking gel buffer: 0.5 M Tris–HCl buffer pH 6.8. Dissolve
6.0 g of Tris in 80 mL of distilled water. Use 4 M HCl to bring
pH value to 6.8 and add distilled water to 100 mL. Store at
4 °C.
4. SDS solution: 10% (w/v) solution in water. Dissolve 10.0 g of
SDS in a total of 100 mL distilled water. Store at room
temperature.
5. Ammonium persulfate: 10% (w/v) solution in water. Dissolve
0.1 g of APS in a total of 1 mL distilled water (see Note 1).
6. N,N,N′,N′-Tetramethyl-ethylenediamine (TEMED).
7. Solution for overlaying running gel during polymerization:
n-butanol saturated with distilled water. Add distilled water to
100 mL of n-butanol until stable layer of water is formed (see
Note 2).
8. SDS-PAGE buffer: 0.025 M Tris–HCl, 0.192 M glycine, 0.1%
SDS, pH 8.3. Dissolve 3.0 g of Tris, 14.4 g of glycine, and
1.0 g of SDS in a total of 1000 mL distilled water. No need for
pH adjustment.
9. Solution buffer (5× concentrate): 60 mM Tris buffer pH 6.8,
25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, and 0.1%
bromphenol blue. Mix 1.2 mL 0.5 M Tris buffer pH 6.8,
5.0 mL glycerol, 2 mL 10% SDS, 0.5 mL 2-mercaptoethanol
(see Note 3), 1.0 mL 1% bromphenol blue, and add water to
10 mL. Store at −20 °C.
10. Staining solution: 0.1% CBB R-250, 50% methanol, 10% acetic
acid. Dissolve 0.5 g of CBB R-250 in 100 mL of methanol
until it is completely without particles. Add 10 mL of acetic
acid and water to 200 mL.
11. Destaining solution: 50% methanol, 10% acetic acid. Mix
500 mL methanol with 100 mL acetic acid and add water to
1000 mL.
12. Gel storage solution: 7% acetic acid. 7 mL of acetic acid in a
total of 100 mL solution.
Novel Collagenolytic Proteases 73
2.2 Collagenolytic 1. The ninhydrin reagent is made by mixing equal volumes of

and Gelatinolytic ninhydrin solution [4% (w/v) ninhydrin in propylene glycol]
Assay (Ninhydrin and 200 mM citrate buffer, 0.16% (w/v) SnCl2, pH 5.0.
Method) Incubate 10 min at 100 °C, after which cool to room tempera-
ture and dilute with 400 μL 50% (v/v) 1-propanol.
2.3 Effect of pH For both the ninhydrin method and the gelatin SDS-PAGE, pre-
on Collagenolytic pare the following buffers:
Activity—
1. 50 mM Na citrate buffer pH 3.0–6.0.
Ninhydrin Method
2. 50 mM Na phosphate buffer pH 6.5 and 7.0.
3. 50 mM Tris–HCl buffer pH 8.0 and 9.0.
4. 50 mM Glycine buffer pH 9.5.
3 Methods
3.1 PAGE 1. Prepare the samples by mixing with the adequate volume of 5×
and Zymography solution buffer and incubating at 95 °C for 5–10 min. For
gelatin SDS-PAGE incubate the samples mixed with 5× solu-
tion buffer at 60 °C for 30 min.
2. Prepare running and stacking gel solutions by mixing previ-
ously prepared solutions according to Tables 1 and 2, respec-
tively (see Note 4).
3. Cast running gel within a gel cassette. Allow space for stacking
gel and overlay immediately with n-butanol solution.
4. Polymerization of the gel is usually completed after 20–30 min.
Pour off n-butanol layer and wash the surface of the gel 3×
with distilled water.
Table 1
Solution volumes for 10 mL of running gel
7.5% 10% 12%

Solution running gel running gel running gel
Monomer solution (mL) 2.50 3.33 4.00
Running gel buffer (mL) 2.50 2.50 2.50
a
Distilled water (mL) 4.84 4.00 3.33
TEMED (μL) 5 5 5
SDS solution (μL) 100 100 100
APS solution(μL) 50 50 50
a
For gelatin SDS-PAGE add 0.3% gelatin solution instead of water
Table 2
Solution volumes for 4% stacking gel
Solution 5 mL 10 mL

Monomer solution (mL) 0.67 1.33
Stacking gel buffer (mL) 1.25 2.50
Distilled water (mL) 3.00 6.00
TEMED (μL) 2.5 5
SDS (μL) 50 100
APS (μL) 25 50
5. Add stacking gel solution. Place the well-forming comb into

the stacking gel. Polymerization is usually completed after
20–30 min. Remove the comb and wash the wells 3× with
distilled water.
6. Fill the upper and bottom tank with SDS-PAGE buffer and
load samples into the wells.
7. Connect the apparatus to the power pack and set to 80 V at the
beginning until samples concentrate and reach the running
gel. Then increase the voltage to 150–180 V and run until
bromophenol blue dye reaches the end of the gel.
8. For the SDS-PAGE, separate the cassette and place the run-
ning gel into the staining solution for constant mixing for
30 min [21].
9. For the gelatin SDS-PAGE, separate the cassette and place the
running gel into distilled water. Wash the gel for 30 min (3×
10 min) to remove SDS. Place the gel into buffer with opti-
mal pH for collagenase activity and incubate overnight at
room temperature. Stain the gel with staining solution for
30 min [22].
10. Destain the gels with destaining solution until clearly visible
blue bands appear on transparent background.
11. Gel can be stored in 7% acetic acid solution.
3.2 Collagenolytic 1. Slowly dissolve collagen or gelatin in 50 mM TBS pH 8.1 (or
and Gelatinolytic buffer with pH optimum for collagenase activity) at 37 °C for
Assay (Ninhydrin 15 min to a concentration of 5 and 20 mg/mL respectively.
Method) 2. Add 10 μL of the sample to 100 μL of the substrate (collagen
or gelatin) and incubate for 5 h at 37 °C with gentle shaking
(see Note 5).
3. Add an equal volume (110 μL) of cold 20% (w/v) solution of
PEG 6600.
4. Incubate with 110 μL 20% (w/v) PEG 6600 at 4 °C for 1 h.
5. Centrifuge at 12,000 × g for 30 min at 4 °C.

6. Add 20 μL of the supernatant to 200 μL ninhydrin reagent (see
Note 6).
7. Measure the absorbance at 570 nm, compare it with the absor-
bance of the blank (ninhydrin reagent and buffer). One arbi-
trary unit of collagen or gelatin digestion activity (CDU and
GDU, respectively) was defined as the amount of enzyme
that releases peptides from collagen or gelatin equivalent in
ninhydrin color to 1 mmol of leucine in 5 h. Calibration
curve with different concentrations of leucine needs to be
constructed [23].
3.3 Effect 1. Prepare the inhibitor and metal ion solutions in adequate con-
of Inhibitors and Metal centration in order to fully inhibit sample collagenase.
Ions on Collagenolytic 2. Preincubate the sample with the various inhibitors or metal
Activity ions at room temperature for 30 min.
3. To test the effect of the inhibitors and metal ions on enzymatic
activity, run the preincubated samples on gelatin SDS-PAGE.
4. After electrophoresis slice the gel into strips containing each
sample and then incubate the slice in a buffer containing the
respective inhibitor or ion overnight.
5. After incubation stain the gel strips with staining solution
(Subheading 2.1). The inhibition is expressed as a percentage
of intensity of the proteolytic bands in the presence of inhibi-
tor compared to a control [24].
3.4 Collagen 1. Add 15 μL of 0.1 mg/mL sample to 60 μL of 0.5 mg/mL col-

Cleavage Assay lagen in buffer with pH optimum for collagenase activity and
incubate for an adequate period of time at 37 °C (see Note 5).
2. Prepare similar reaction mixtures and preincubate with ade-
quate concentration of inhibitors.
3. Run the samples on 7.5% SDS-PAGE gel. Observe collagen
degradation products [2].
3.5 Effect of pH 1. Prepare 2% gelatin in the various buffers.

on Collagenolytic 2. Incubate the sample in the various 2% gelatin solutions for 5 h,
Activity— after which determine the enzyme activity as described in
Ninhydrin Method Subheading 2.2 (starting from item 3).
3.6 Effect of pH 1. Resolve collagenase on gelatin SDS-PAGE as described in

on Collagenolytic Subheading 2.1.
Activity—Gelatin 2. After electrophoresis (and after washing the gel 3× with distilled
SDS-PAGE water) slice the gel into strips containing sample triplicates and
then incubate each strip in a previously prepared buffers of vari-
ous pH values overnight.
3. After incubation stain the gel strips with CBB. The relative
activity is expressed as a percentage of intensity of the most
intensive proteolytic bands.
3.7 Effect of pH 1. Prepare the following buffers: 50 mM Na citrate buffer
on Collagenolytic pH 3.0–6.0, 50 mM Na phosphate buffer pH 6.5 and 7.0,
Stability— 50 mM Tris–HCl buffer pH 8.0 and 9.0 and 50 mM glycine
Ninhydrin Method buffer pH 9.5.
2. Preincubate collagenase in previously prepared buffers of vari-
ous pH values for 30 min.
3. Prepare 2% gelatin solution in 0.5 M TBS pH 8.1 (or buffer
with pH optimum for collagenase activity).
4. Determine the enzyme activity as described in Subheading 2.2
(starting from the item 2).
3.8 Effect of pH 1. Prepare the following buffers: 50 mM Na citrate buffer
on Collagenolytic pH 3.0–6.0, 50 mM Na phosphate buffer pH 6.5 and 7.0,
Stability—Gelatin 50 mM Tris–HCl buffer pH 8.0 and 9.0 and 50 mM Glycine
SDS-PAGE buffer pH 9.5.
2. Preincubate collagenase in previously prepared buffers of vari-
ous pH values for 30 min.
3. Resolve collagenase on gelatin SDS-PAGE as described in
Subheading 2.1.
4. After electrophoresis (and after washing the gel 3× with dis-
tilled water) incubate the gel in the buffer optimal for collage-
nase activity overnight.
5. After incubation stain the gel with CBB. The relative activity is
expressed as a percentage of intensity of the most intensive
proteolytic bands.
3.9 Effect 1. To determine the optimal temperature for enzyme activity,

of Temperature incubate the reaction mixtures at various temperatures (20–
on Collagenolytic 80 °C) for 10 min.
Activity 2. Determine the enzyme activity as described in Subheading 2.2.
3. The residual activity was expressed as a percentage of maximal
activity.
3.10 Effect 1. To determine the thermostability of the enzyme, preincubate

of Temperature the reaction mixture at a given temperature between 20 and
on Collagenolytic 90 °C for 30 min, and then determine the enzyme activity at
Stability 37 °C after incubation as described in Subheading 2.2.
2. The residual activity was calculated as the ratio between the
enzymatic activity, observed at the end of each incubation run,
and that at the beginning, and expressed as a percentage of
maximal activity [24].
4 Notes
1. It is recommended to prepare this solution immediately before

usage.
2. Mix the solution before usage and wait for the layers to sepa-
rate. Use upper layer.
3. For electrophoresis under nonreducing conditions add equal
volume of water instead of 2-mercaptoethanol.
4. Solution of APS needs to be added immediately before casting
the gel.
5. The amounts of collagenase and substrate need to be
optimized.
6. Ninhydrin reagent needs to be made immediately before usage.
References
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7:728–735 2, Unit2.1:2.1.1–2.1.12
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(2007) Plant collagenase L unique collageno- K, Ohshima H (1995) Use of standardized pro-
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cum collagenase. Biochemistry 49:5314–5320 CA, Hansbrough WB, Dore C (1995) Wound
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Tallis A, Motley TA, Wunderlich RP,
5. Hu X, Beeton C (2010) Detection of func- Dickerson JE Jr, Waycaster C, Slade HB
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of beef with bacterial collagenase. Meat Sci genase: a novel strategy for arrhythmia therapy.
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NK, Sadulla S (2008) Studies on the influence (2007) Factors influencing the collagenase
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Chapter 8
Zymography as a Research Tool in the Study of Matrix

Metalloproteinase Inhibitors
Zongli Ren, Juanjuan Chen, and Raouf A. Khalil
Abstract
Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade various components of the
extracellular matrix (ECM) and play a role in tissue remodeling. Changes in MMPs have been observed
in cancer, connective tissue disorders, and vascular disease, and both endogenous tissue inhibitors of
MMPs (TIMPs) and synthetic MMP inhibitors (MMPIs) have been evaluated as modulators of MMP
activity in various biological systems. Zymography is a simple technique that is commonly used to assess
MMP activity and the efficacy of MMPIs. Also, reverse zymography is a modified technique to study the
activity of endogenous TIMPs. However, problems are often encountered during the zymography pro-
cedure, which could interfere with accurate assessment of MMP activity in control specimens, and thus
make it difficult to determine the pathological changes in MMPs and their responsiveness to MMPIs.
Simplified protocols for preparation of experimental solutions, tissue preparation, regular and reverse
zymography procedures, and zymogram analysis are presented. Additional helpful tips to troubleshoot
problems in the zymography technique and to enhance the quality of the zymograms should make it
more feasible to determine the changes in MMPs and assess the efficacy of MMPIs in modulating MMP
activity in various biological systems and pathological conditions.
Key words Blood vessels, Uterus, Extracellular matrix, Matrix metalloproteinase, TIMP
Abbreviations
A/C Acrylamide/bis-acrylamide
ECM Extracellular matrix
MMP Matrix metalloproteinase
MMPI MMP inhibitor
MT-MMP Membrane-type-MMP
RUPP Reduction in uterine perfusion pressure
TIMP Tissue inhibitor of MMP
Zn
2+
Zinc ion
79
80 Zongli Ren et al.
1 Introduction
Matrix metalloproteinases (MMPs) are structurally and function-

ally related Ca2+-dependent and Zn2+-containing endopeptidases
that degrade the extracellular matrix (ECM) and connective tissue
proteins [1–3]. Since its discovery, the MMP family has grown to
28 members in vertebrates, at least 23 in humans and 14 in blood
vessels [2]. Based on their activity toward specific substrates and
the organization of their different domains, MMPs are classified
into collagenases, gelatinases, stromelysins, matrilysins, membrane-
type (MT)-MMPs, and other MMPs [4–6]. MMPs are secreted as
inactive or latent pro-MMPs which undergo proteolytic activation
by other MMPs or other proteases before they can degrade ECM
proteins [7] (Fig. 1). Activated MMPs play a role in vascular
remodeling and angiogenesis [1], the uterine and vascular changes
associated with pregnancy and preeclampsia [8], and many patho-
logical conditions such as neoplasm, connective tissue disorders,
and vascular disease.
Because there are no specific activators of MMPs, MMP inhib-
itors (MMPIs) are often used to test the role of MMPs in different
processes. Also, in many tissues, MMP activity is modulated by
endogenous tissue inhibitors of metalloproteinases (TIMPs) [9,
10] (Fig. 1). TIMPs include four homologous members; TIMP-1,
-2, -3, and -4 [11–13]. While all TIMPs can inhibit all MMPs, the
efficacy of MMP inhibition varies with each TIMP. For example,
TIMP-1 is a poor inhibitor of membrane-type 1-MMP (MT1-
MMP), MT3-MMP, MT5-MMP, and MMP-19 [12]. Also,
TIMP-1 and -3 interact with pro-MMP-9, while TIMP-2, -3, and
-4 interact with pro-MMP-2 [12]. Although TIMPs could restrict
ECM deposition, their ultimate effect on ECM turnover depends
on the TIMP/MMP ratio in the tissue. Other endogenous MMPIs
include α2-macroglobulin, a glycoprotein consisting of four iden-
tical subunits that is found in blood and tissue fluids and acts as a
general proteinase inhibitor. Most endopepidases are inhibited by
being entrapped within the macroglobulin and the complex is then
cleared by endocytosis via a low-density lipoprotein receptor-
related protein-1 [14].
In addition to endogenous MMPIs, several classes of synthetic
MMPIs have been developed and evaluated as diagnostic and thera-
peutic tools in cancer, autoimmune, and vascular disease [15]
(Table 1). Early investigations on MMPIs focused on developing
compounds that contain a group that chelates the catalytic Zn2+ ion
and a backbone that mimics the natural peptide substrate of MMPs
[16]. The first-generation hydroxamate-based MMPIs have the
Zn2+-binding group hydroxamate and the basic backbone of colla-
gen [17]. Batimastat, a low molecular mass hydroxamate derivative,
was the first MMPI to enter clinical trials [18], but the results were
Zymography and MMP Inhibitors 81
NH2
Zn2+ Zn2+
B C
S-O-SG
GSH
Oxidative Stress COO-
Inter and Intra NH3+
ONOO- Molecular
Proteolysis NH2 Substrate NH2
Michaelis
NH2
A SH Complex
Zn2+ Zn2+ Zn2+

Proteolytic Activation
Other MMPs
Pro-MMP Active MMP

Substrate
TIMP
Hyroxamate-Based
Non-hydroxamate-Based
NH2 NH2 Zn2+-Binding MMPIs
TIMP
D Zn2+ E Zn2+
Inactive MMP Inactive MMP
Fig. 1 Mechanisms of MMP activation, MMP–substrate interaction, and MMP inhibition. Full-length pro-MMP
can be activated in two ways. (a) Proteolytic activation of MMPs by MT-MMP/TIMP or other proteases occurs
by removal of the cysteine switch motif-SH autoinhibitory propeptide region resulting in a truncated active
MMP. (b) In the presence of oxidative stress, reactive O2 species such as peroxinitrite (ONOO−) and cellular
glutathione (GSH), the critical cysteine residue in the propeptide region, undergoes S-glutathiolation, leading
to the release of cysteine binding to the catalytic Zn2+ ion and active enzyme. (c) Active MMP interacts with its
substrate through a series of biochemical reactions. Using H+ from free H2O, the substrate carbonyl binds to
Zn2+, forming a Michaelis complex. The Zn2+-bound H2O performs a nucleophilic attack on the substrate,
resulting in the release of an H2O molecule, breakdown of the substrate, and the release of active MMP to be
ready for interaction with another substrate. (d) TIMP interacts with MMP in a manner similar to that of sub-
strate substituent, further contributing to expelling Zn2+-bound H2O and preventing substrate degradation. (e)
Zn2+-binding MMPIs act as anchor that is locked in the active site and direct the backbone of the inhibitor into
the target substrate-binding pockets resulting in inactive MMP. Dashed lines indicate inhibition
disappointing due to the metabolically labile nature of the hydroxa-

mate Zn2+-binding group, the metabolic inactivation and chelation
of metal ions of other metalloproteins, and the serious side effects
such as musculoskeletal pain experienced by patients [19].
Non-hydroxamate-based Zn2+-binding MMPIs such as car-
boxylates, hydrocarboxylates, sulphydryls, phosphoric acid
derivatives, and hydantoins are more stable and do not have the
limitations associated with hydoxamate-based MMPIs. Rebimastat
is a broad-spectrum MMPI that has a thiol Zn2+-binding group
82
Table 1
Representative synthetic MMPIs and their IC50 or Ki toward specific MMPs
MMP specificity (IC50 or Ki) (nM)

Zongli Ren et al.
MMPIa
(other name) MMP-1 2 3 7 8 9 10 11 13 14 16
Hydroxamate-based 3 4 20 6 10 1
Batimastat
(BB-94)
Marimastat 5 6 200 20 2 3 1.8
(BB-2516)
Ilomastat 0.4 0.4 27 0.1 0.2 5.2
(GM6001)
CGS-27023-A 33 20 43 8 8 6
(MMI-270)
MMI-166 0.4 400 90 100
ABT-770 4600 3.7 42 >10,000 120
PD-166793 6.1 47 12 7.2 7.9 8 240
Prinomastat 8.3 0.05 0.3 54 0.26 0.03 0.33
(AG3340)
Cipemastat 3.0 154 4.4 59 3.4
(Ro 32-3555)
Non-hydroxamate 9 39 157 23 27 40
Rebimastat
(BMS-275291)
Tanomastat 11 134 301 1470
(BAY 12-9566)
Mechanism-Based 206 14 15 96 600
SB-3CT
Pyrimidine-based 16,000 7–246 1200 15 12–23 96 91
Ro 28-2653
Phosphorous-based 2000 55 20,000 4 41 45 6.2 16 90
RXP03
Tetracyclines >400,000 56,000 32,000 28,000 26,000– 2000–50,000
Doxycycline 50,000
Metastat 34 48 μg/mL 0.3 μg/mL
(COL-3;CMT-3) μg/mL
Antibody-based 0.8
DX-2400 (Ki)
REGA-3G12 +++
a
MMPI MMP inhibitor, CMT chemically modified tetracycline, IC50 half-maximal inhibitory concentration, Ki inhibition constant
Zymography and MMP Inhibitors
83
[20, 21]. Tanomastat has a thioether Zn2+-binding group and a

biphenyl deep-pocket-binding segment and is well-tolerated, but
may show variable efficacies and outcomes depending on the tim-
ing of administration [22].
Mechanism-based MMPIs bind to the MMP active site and
cause covalent enzyme modification. SB-3CT, a thiol-based inhibi-
tor that contains a diphenylether deep-pocket-binding scaffold, is
a mechanism-based selective inhibitor of MMP-2 and -9 through a
process involving slow-binding inhibition similar to that of TIMP-1
and -2 [16, 23, 24]. SB-3CT directly binds the catalytic Zn2+ ion
of MMP-2 and changes the conformation around the Zn2+ active
site to that of the proenzyme [25].
Pyrimidine-based MMPIs include Ro 28-2653, an orally bio-
available MMPI that inhibits MT1-MMP, MT3-MMP, and MMP-
2, -8, and -9 [26, 27]. Phosphorous-based MMPIs have
phosphinate as Zn2+-binding group and include 582311-81-7 and
the MMP11-selective inhibitor RXP03 [28, 29]. Tetracyclines,
such as minocycline and doxycycline, have innate MMPI capacity.
Doxycycline is the only MMPI approved by the United States
Food and Drug Administration and is indicated for periodontal
disease [30]. Metastat (COL-3) is a chemically modified tetracy-
cline that has been tested in a Phase I clinical trial in patients with
refractory metastatic cancer [31].
Because of the high structural homology of the MMP catalytic
site, most of the early MMPIs show broad-spectrum effects on dif-
ferent MMPs. In order to reduce off-target effects of MMPIs,
investigations have shifted from targeting the catalytic site to alter-
native sites in the MMP molecule. MMPs have unprimed subsites
S1, S2, and S3 on the left side of the Zn2+ ion and primed S1′, S2′,
and S3′ on the right side of Zn2+ ion [32, 33]. The S1′ pocket is
the main substrate recognition subsite and is the most variable
among different MMPs in terms of amino acid sequence and
pocket depth (shallow, intermediate, and deep) [16, 34–36]. These
variabilities in MMP structure have been utilized to design more
specific MMPIs. For example, extending the P1′ substituent (the
group in MMPI or substrate that binds to the S1′ pocket of MMP)
was used to enhance selectivity of MMP-13 over the highly homol-
ogous MMP-2 by taking advantage of the steric limitations of the
shorter S1′ loop of MMP-2 [37]. However, identifying alternative
MMP-specific sites could be challenging as they are scattered in
different locations on the surface and even hidden inside the MMP
molecule. Combining structural spectroscopic analyses, nuclear
magnetic resonance, and protein crystallography with computa-
tional prediction of binding sites have helped to reveal these h idden
sites and made it possible to design novel molecular effectors and
therapeutic agents [38, 39]. For example, peptide-based MMPIs
interact with secondary binding sites on MMPs and thereby have
greater selectivity [40]. Also, phage display peptide libraries have
been used to identify selective MMP-2, MMP-9, and MT1-MMP

inhibitors that are effective in vivo [41, 42].
Antibody-based MMPIs have been designed to achieve high
selectivity and potency [43]. For example, combining a human
antibody phage display library with automated selection and
screening strategies resulted in the identification of the highly
selective antibody-based MMP14 inhibitor DX-2400 [44, 45].
Also, the neutralizing monoclonal antibody REGA-3G12 is a
selective inhibitor of MMP-9 directed against the catalytic domain,
but not the fibronectin or Zn2+-binding domains [46, 47]. Another
strategy for generating inhibitory antibodies that effectively target
the in vivo activity of dysregulated MMPs is mimicking the mecha-
nism used by TIMPs [48].
While several MMPIs have been developed, only one MMPI is
approved by the Food and Drug Administration [1]. This could be
due to the numerous side effects of MMPIs and their lack of speci-
ficity in various MMP assays. In order to determine the role of
MMPs in pathological conditions and the efficacy of various
MMPIs, it is critical to have reliable methods for measuring MMP
activity. Zymography is a simple technique first described in 1980
[49] and is now widely used to measure MMP activity in various
systems. The zymography technique is based on separation of pro-
teins by nonreducing sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS–PAGE). Special polyacrylamide gel is made
by inducing acrylamide polymerization in the presence of the spe-
cific substrate of the MMP(s) of interest. For instance, using gela-
tin as a substrate, zymography has been used to assess the activity
of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) [50]. During
electrophoresis, MMPs are activated in a nonproteolytic manner
by SDS [51]. Following electrophoretic separation and a “renatur-
ation” step, the gel is incubated at 37 °C in a Ca2+- and Zn2+-
containing buffer optimized for measuring MMP activity toward
the specific substrate (Fig. 2).
If performed carefully and analyzed accurately, zymography
can be a very valuable technique to measure MMP activity in vari-
ous biological systems including plasma, cells, culture media, and
tissue extracts [52, 53]. Zymography has also been used to mea-
sure the changes in MMP activity in vascular remodeling and vas-
cular disorders [3]. Also, reverse zymography is a modified
technique to study the activity of endogenous TIMPs. However,
problems encountered during the zymography procedure may lead
to inaccurate assessment of MMP activity in control specimen, and
thus make it difficult to determine the pathological changes in
MMP activity and their responsiveness to MMPIs.
In this chapter, we will discuss the materials needed for a good
zymography experiment, the preparation of experimental buffers,
sample preparation, running and developing the gels during the
regular and reverse zymography procedures, and zymogram analysis
Gelatin Zymography Procedure
Tissue Dissection Tissue

Sample Preparation
Tissue Grinder
Tissue Homogenization
Centrifuge 10000 x g for 2 min
10 to 25 ml Marker
Marker and Sample Loading in Gel
Polyacrylamide Gel
Running the Gel
Electrophoresis
- SDS, +Triton X-100

Gel Renaturing and Developing
CaCl2, ZnCl2
Coomassie Blue R-250

Gel Staining and Destaining
Methanol:Acetic Acid:dH2O
50 : 10 : 40
ImageJ
Imaging and Zymograph Analysis MMP
Actin
Pixel Intensity x Band Area in mm2
Fig. 2 Flow chart of gelatin zymography procedure
and interpretation. We will also provide helpful notes to trouble-

shoot problems and some tips to enhance the quality of the zymo-
grams and assess the efficacy of MMPIs.
2 Materials
2.1 Materials The following materials should be prepared before starting a

for Zymography zymography experiment. All materials should be clean and stock
Experiment solutions should be clear (see Note 1).
1. Glass plates with 0.75-mm-thick spacers and ten well combs.
2. 30% w/v Acrylamide/bis-acrylamide (A/C).
3. Separating gel buffer stock: 1.5 M Tris–HCl, pH 8.8. Use
27.23 g Tris-base in 150 mL dH2O, adjust pH to 8.8 with 38%
HCl.
4. Stacking gel buffer stock: 0.5 M Tris–HCl, pH 6.8. Use 6 g
Tris-base in 100 mL dH2O, adjust pH to 6.8 with 38% HCl.
5. 1% w/v gelatin. Use 100 mg gelatin in 10 mL dH2O. Warm
the solution to 60 °C in a water bath and vortex repeatedly
until it becomes almost translucent (~20 min). Cool down the
gelatin solution to room temperature before use.
6. 10% w/v SDS. Use 1 g SDS in 10 mL dH2O.
7. 10% w/v ammonium persulfate (APS). Use 1 g APS in 10 mL
dH2O.
8. N,N,N′,N′-Tetramethylethylenediamine (TEMED).
9. Running buffer stock: For 1× running buffer, use 0.024 M or
2.9 g Tris-base, 0.192 M or 14.4 g glycine, and 0.1% w/v or
1 g SDS in 1 L dH2O, pH 8.3. For 10× running buffer, use
0.24 M or 29 g Tris-base, 1.92 M or 144 g glycine and 1% w/v
or 10 g SDS in 1 L dH2O, pH 8.3. Remember to dilute the
10× running buffer 1:10 in dH2O before use.
10. Sample buffer (2×): Use 2.5 mL of 0.5 M Tris/HCl pH 6.8,
2 mL glycerol, 4 mL of 10% w/v SDS (1 g SDS in 10 mL
dH2O), and 0.5 mL of 0.1% w/v bromophenol blue (0.001 g
in 1 mL dH2O). Add dH2O to 10 mL. When ready to load the
samples in the gel and depending on the protein concentration
in the sample tissue homogenate, add one part of the sample to
one part sample buffer.
11. Renaturing buffer. For 1× renaturing buffer (Triton X-100
2.5% v/v), add 5 mL Triton X-100 in 195 mL deionized
dH2O. For 10× solution stock (Triton X-100 25% v/v), add
25 mL Triton X-100 in 75 mL deionized dH2O.
12. Developing buffer (1×): 50 mM or 6.06 g Tris-base, 0.2 M or
11.688 g NaCl, 5 mM or 0.555 g CaCl2, 0.02% w/v or 0.2 g
Brij35, and 1 μM or 0.136 mg ZnCl2 in 1 L dH2O, pH 7.6.
13. Staining solution: 0.5% w/v or 2 g Coomassie blue R-250,
25% v/v or 100 mL isopropanol, and 10% v/v or 40 mL acetic
acid to 260 mL dH2O. Filter through filter paper before use.
14. Destaining solution: Add methanol:acetic acid:dH2O in the

following proportion, 50:10:40. Final concentration is 50%
(v/v) methanol and 10% (v/v) acetic acid in dH2O.
15. Homogenization buffer: 0.02 M or 0.418 g 3-[N-Morpholino]
propanesulfonicacid (MOPS), 4% w/v or 4 g SDS, 10% v/v or
10 mL glycerol, and 90 mL dH2O. Immediately before use,
add the following solutions: 1.5 mL homogenization buffer,
35 μL of 50 mM ethylenediaminetetraacetic acid (EDTA), and
75 μL of 20× anti-protease cocktail.
16. Anti-protease cocktail (20×): To make 1.5 mL stock, use 0.4%
w/v bovine serum albumin (BSA) (take 0.3 mL of 2% w/v or
2 g BSA in 100 mL dH2O), 0.11 mM or 165 μL of 1 mM
leupeptin, 0.11 mM or 165 μL of 1 mM pepstatin, 0.15 TIU
or 60 μL of 7.6 TIU/mL aprotinin, 0.4 mM or 60 μL of
10 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochlo-
ride (AEBSF), and 0.1 M or 750 μL of 200 mM ethylene gly-
col tetraacetic acid (EGTA).
2.2 Materials 1. Prepare all materials as for regular zymography.

for Studying MMPIs 2. Prepare stock solution for the MMPI of interest. For example,
Using Zymography stock solution (10−2 M) of SB-3CT (MMP-2/MMP-9 inhibi-
tor IV), Ro-28-2653 (5-biphenyl-4-yl-5-[4-(-nitro-phenyl)-
piperazin-1-yl]-pyrimidine-2,4,6-trione), and Batimastat
(BB-94) is prepared in dimethylsulfoxide (DMSO) [54].
2.3 Materials 1. Prepare all materials as for regular zymography.

for Reverse 2. Prepare human recombinant MMP-2 or MMP-9 (0.13 μg/mL).
Zymography
3 Methods
3.1 Gel Preparation 1. Prepare 8% A/C separating gel (for two 0.75 mm gels) (see
Note 2): 4.6 mL dH2O, 1 mL 1% w/v gelatin (100 mg/10
mL) (see Note 3), 3.0 mL 1.5 M Tris/HCl (pH 8.8), 3.2 mL
30% A/C stock, 120 μL 10% w/v SDS (1 g/10 mL), 120 μL
10% w/v APS (1 g/10 mL).
2. Prepare 5% A/C stacking gel: 3.6 mL dH2O, 1.56 mL 0.5 M
Tris/HCl (pH 6.8), 0.6 mL 30% A/C stock, 20 μL 10% w/v
SDS (1 g/10 mL), 140 μl 10% w/v APS (1 g/10 mL).
3. TEMED will polymerize the gel and should be added last to
the freshly prepared separating and stacking gel, immediately
before pouring into the cassette. Add 5 μL of TEMED to the
separating gel solution to initiate the polymerization process.
Swirl the solution rapidly and avoid bubble formation. Pipette
3 mL of the separating gel solution into each cassette, avoiding
the formation of bubbles. Carefully, overlay the separating gel

with dH2O, filling up to the top of the cassette. Do not disturb
the surface of the separating gel solution. Allow the gel to
polymerize for at least 30 min at room temperature. The
warmer the condition the gel is in, the less the polymerization
time. Do not touch the gels until polymerization is complete
as indicated by a clear straight line of separation between the
gel phase and the water phase.
4. Decant the water from the top of the separating gel and keep
the gel upside down on bench top for a few minutes to drain
away all the water. Use filter paper to absorb residual water.
5. Add 10 μL of TEMED to the stacking gel solution, swirl
rapidly, and immediately pipette the solution on top of the
already polymerized separating gel until it reaches the top
of the front glass plate. Rapidly insert the appropriate comb
(ten wells or lanes) into the liquid stacking gel and ensure
that no bubbles are trapped under the comb. Allow the
stacking gel to polymerize at room temperature (about
30 min). Place the gels (comb on top) in a sealed plastic
bag or a container having 1× running buffer to keep them
moist. The gels can be stored at 4 °C for up to 2–3 weeks
before running.
3.2 Sample 1. Preparation of the biological sample is critical for a success-

Preparation ful zymography. It is relatively easy to detect MMP activity
in culture media and cell lysates. However, analysis of
MMPs in tissues such as the aorta or uterus could be diffi-
cult (see Note 4).
2. Weigh approximately 50 mg of the tissue of interest. Cut the
tissue into small pieces in a small weighing boat on ice. Transfer
the tissue to a tissue grinder or mortar (Kimble) on ice. Add
cold homogenization buffer with anti-protease cocktail (for
50 mg tissue add 300 μL homogenization buffer). The volume
of homogenization buffer may vary depending on the tissue
used. Homogenize the tissue completely using the tissue
grinder or mortar and pestle (Kimble) on ice.
3.
Transfer homogenate to labeled microcentrifuge tubes.
Centrifuge the homogenate at 10,000 × g for 2 min at 4 °C. If
the tissue homogenate contains floating lipids, repeat centrifu-
gation at least two times to obtain a clear supernatant. Save the
supernatant, discard the pellet, and measure the protein con-
centration in the supernatant using Bradford protein assay
[55]. Before loading, adjust the protein concentration in the
samples using sample buffer. Ideally, mix one part sample with
one part sample buffer and let stand at room temperature for
at least 30 min to allow coating of the protein with SDS. DO
NOT HEAT.
3.3 Running the Gel 1. Gently pull the comb out of the stacking gel and peel off the
and Electrophoresis rubber band at the bottom of the cassette.
2. Gently place the cassette in the gel protean II apparatus (Bio-
Rad). Fill the buffer chambers with 1× running buffer. Reducing
agents such as dithiothreitol (DDT) are omitted because of
possible interference with subsequent refolding of gelatinases.
Load the samples in the gel lanes. Typically, 10–25 μL of each
sample which contains 0.1–10 μg of the enzyme is loaded to
each of the gel lanes (see Note 5). Molecular weight markers are
included on each gel preferably in lanes 1 and 6.
3. Run the gel in the protean II apparatus (Bio-Rad) using gel
electrophoresis at the standard running conditions (125 V,
constant voltage) and until the bromophenol blue tracking dye
reaches the bottom of the gel (see Notes 6 and 7). Running
time could range between 60–120 min depending on the gel
A/C percentage, running buffer concentration, and pH. The
proteins will be separated according to their molecular weight;
whereby the low molecular weight proteins will run faster and
farther than the high molecular weight proteins which will be
lagging behind (Fig. 2).
3.4 Renaturing, 1. Dilute the renaturing buffer (10×) 1:10 with deionized dH2O
Developing, Staining, to obtain 1× renaturing buffer solution. Carefully remove the
and Destaining the Gel gel from the cassette and place it in a plastic tray containing 1×
renaturing solution (50 mL for one mini-gel). Incubate the gel
for 30 min at room temperature with gentle shaking in order
to remove SDS which causes MMPs to denature and become
inactive (see Notes 8 and 9).
2. Decant the zymogram 1× renaturing buffer and replace with
fresh 1× developing buffer (50 mL for one mini-gel). Equilibrate
the gel at room temperature for an additional 30 min in the
developing buffer with gentle shaking. Decant the developing
buffer and replace with 50 mL of fresh 1× developing buffer and
incubate the gel at 37 °C overnight for ~18 h for maximum sen-
sitivity (see Note 10). Optimal results are determined empiri-
cally by varying the sample load and incubation time (Fig. 3).
3. Decant the developing buffer and stain the gel with Commassie
blue R-250 staining solution for at least 30 min until the gel is
uniformly dark blue. Destain the gel with destaining solution
until areas of gelatinolytic activity appear as clear sharp bands
against dark blue background.
3.5 Zymogram The goal of gelatin zymography is to obtain clear and sharp bands
Analysis of the digested substrate against a blue background of the unde-
graded substrate (Fig. 3). Comparison of the location of the gelati-
nolytic with molecular weight standards run simultaneously on the
Fig. 3 Concentration-dependent MMP-2 and MMP-9 gelatinase activity in uterus of pregnant rats. Uterine tis-
sue strips from normal pregnant rats were homogenized and prepared for gelatin zymography analysis using
different concentrations of loaded protein (0.1–20 μg). Pro-MMP-2, MMP-2, pro-MMP-9, and MMP-9 showed
concentration-dependent gelatinolytic bands because of the presence of their preferred substrate gelatin.
Other MMPs are only detected at higher protein concentration and are less clear because gelatin is not their
preferred substrate
same gel should help identify the specific MMP involved (see Note
11). The bands in the gel are quantified using ImageJ 1.38X
(NIH). The gel image is scaled using a grey scale such that the
intensity of each pixel would range from 0 to 255. The integrated
intensity of the band of interest is calculated by first outlining and
measuring the band area in pixels, then transferring it into mm2
using a calibration bar. The total pixel intensity is measured by
summing the pixel values within the band area, and the average
pixel intensity is measured by dividing the total pixel intensity by
the number of pixels. The average pixel density of the background
is then subtracted from the average band intensity. The integrated
intensity of the selected band is then measured as average pixel
intensity × band area in mm2 [54, 56]. The integrated intensity of
the band can also be normalized to the housekeeping protein actin
to correct for loading. Comparison of the integrated intensity of
the bands would help determine the changes in MMP activity in
specimens in different physiological and pathological conditions
3.6 Study of MMPI 1. Prepare all the materials and follow all the methods as described
above for gelatin zymography.
2. For sample preparation, incubate the sample overnight in phys-
iological buffer solution in the presence of appropriate concen-
tration of MMPI. For example, SB-3CT (MMP-2/MMP-9
inhibitor IV, 10−6 M), BB-94 (10−6 M), or Ro-28-2653
(10−6 M) (see Note 14).
3. Compare the integrated intensity of the gelatinolytic bands in

control samples in the absence of MMPI with that in the pres-
ence of MMPI. The integrated intensity of the gelatinolytic
bands should be less in the presence of MMPI compared to the
control samples in the absence of MMPI.
3.7 Reverse 1. Prepare all the materials and follow the methods as described
Zymography above for regular gelatin zymography.
2. When preparing the separating gel, make the 8% separating gel
as in regular gelatin zymography, but add human recombinant
MMP-2 or MMP-9 (0.13 μg/mL) (see Note 15).
3. For the staining step, decant the developing buffer and stain
the gel with Coomassie Blue R-250 staining solution for at
least 30 min until the areas representing the undigested gelatin
due to the presence of endogenous TIMPs appear as clear dark
blue bands against faint blue background of digested gelatin
caused by the added MMP-2 or MMP-9.
4 Notes
Although zymography could be a sensitive and quantifiable assay

to analyze MMP activity, problems related to the nature, source,
and preparation of samples, the substrate in the gel, and the dis-
tinction between inactive and active forms of MMPs could com-
promise the validity of the technique and complicate interpretation
of the results. In order to obtain consistent and reliable z ymograms,
it is important to pay attention to all the different steps including
the gel preparation, sample preparation, running and developing
the gels, and analysis of the zymograms.
1. For gel electrophoresis, all stock solutions should be prepared
using electrophoresis-grade reagents and kept fresh. If any pre-
cipitations are observed, the quality of the solution and the
concentration of the different ingredients are likely altered and
should not be used. Also, avoid bacterial contamination of the
buffers and solutions as bacterial proteases may result in the
appearance of nonspecific bands. This can be minimized by
filter sterilization of the buffers and stock solutions and storage
at 4 °C.
2. An 8% polyacrylamide gel is generally used for separating gela-
tinases. However, the percentage of acrylamide and the thick-
ness of the separating gel may vary depending on the MMP
type, form, and molecular mass. For instance, to better visual-
ize the dimeric form of MMP-9 (~200 kDa) or to obtain a
better resolution of bands with close molecular weights (latent
and active forms), a lower 4–6% polyacrylamide solution
should be used. However, by taking this approach the gelati-

nolytic bands may become less sharp. On the other hand, to
better visualize the lower molecular weight MMPs such as
MMP-1 and -7, a higher 10–12% polyacrylamide solution can
be used. The gels can be prepared in advance and stored at
4 °C for 2 or 3 weeks without significant effects on the
resolution.
3. Gelatin is commonly used as the protein substrate because it is
inexpensive, easily hydrolyzed by several peptidases, and does
not tend to migrate out of the resolving gel in electrophoretic
tests performed at 4 °C [57]. Gelatin is the substrate of choice
to detect the gelatinases MMP-2 and MMP-9. Other MMPs
such as MMP-1, MMP-8, and MMP-13 can degrade gelatin;
however, the gelatinolytic bands will be weak because gelatin is
not their preferred substrate [58, 59]. For improved detection
of these MMPs, modified zymography has been developed by
incorporating a more suitable substrate such as casein or col-
lagen into the gel, or by enhancing the gelatinolytic signal with
the addition of heparin to the samples [60–62].
4. Fresh tissues should be used for measuring MMP activity, and
the whole procedure of tissue preparation and homogeniza-
tion should be performed on ice at 4 °C. Because of inherent
differences in tissue structure, protein extractability may vary
between and within specimens [63]. For example, tissues such
as the liver and placenta are easy to cut and homogenize due to
their fragile nature, while the aorta and uterus are more diffi-
cult to cut into small pieces and to homogenize because of
their large content of collagen and elastin fibers. The homog-
enized samples should not be boiled as high temperature
causes protein precipitation of the enzymes. Also, reducing
agents such as DTT or 2-mercaptoethanol should not be
added as reducing agents break the disulfide bond and thereby
prevent some forms of tertiary protein folding, inhibit MMP
refolding after electrophoresis [53], and break up quaternary
protein structure in oligomeric subunits. The tissue extraction
procedure itself may activate MMPs or cause inhibition of
active enzymes by interacting with some of the components of
the homogenization buffer. Some studies suggest that EDTA,
other Zn2+ chelators, and protease inhibitors should not be
added to the tissue extract [64]. While EDTA may prevent
MMP activation by binding with the Ca2+ and Mg2+ ion in the
homogenization buffer, MMPs are reactivated after incubation
in the developing buffer which contains Ca2+ and Zn2+ ions.
5. The amount of sample loaded in the gel is critical for successful
zymography as large amounts of tissue extracts may produce
saturated and distorted bands. The sensitivity of zymography is
much greater than that of Western blots, which depends on the
antibody affinity for MMPs. Because zymography is a sensitive

technique, gelatinolytic bands can be detected with MMP lev-
els as low as 10 pg [56, 65]. However, these low MMP levels
are not often detected because the ratio of MMPs to total pro-
tein in crude samples is extremely low. This may make it neces-
sary to load larger amounts of the tissue extract. Overloading
of total protein extracts into the wells or lanes may lead to satu-
rated gelatinolytic bands in the zymogram. For instance, in
pregnant rat uterus extracts, gelatin zymography using differ-
ent protein amounts showed dose- dependent increases in
MMP-2 and MMP-9 proteolytic activity at 0.1, 0.2, and
0.5 μg, clearly discernible bands at 1–2 μg, but saturated bands
at 5, 10, and 20 μg (Fig. 3).
6. For the proteins to move from the cathode to the anode
through the gel, the gel system and running buffer should
have the proper pH as it may affect the mobility of the differ-
ent components of the gel system relative to the proteins. For
example, depending on the pH, glycine can exist in three dif-
ferent charge states; positive, neutral, or negative. Control of
the charge of glycine in the different buffers is key to the
mobility of proteins in the gel. All of the proteins in the gel
have an electrophoretic mobility that is intermediate between
the mobility of glycine and Cl−, so that as the glycine and Cl−
fronts sweep through the sample well, the proteins are concen-
trated in the narrow zone between the two fronts. This process
continues through the stacking gel until the proteins hit the
separating gel, where the pH switches to 8.8. At this pH, the
glycine becomes mostly negatively charged and migrates much
faster than the proteins. As the glycine front accelerates past
the proteins, the proteins become concentrated in a very nar-
row region at the interface of the stacking and separating gels.
Because the separating gel has a greater acrylamide concentra-
tion, it slows the mobility of the proteins according to their
molecular weight and the protein separation begins [66]. If
any of the buffers or gel system pH is altered, the protein
mobility will be affected in areas of the gel with “improper”
pH. This explains the odd protein migration behavior and the
distorted shape of the protein bands in some gels.
7. Careful attention to the temperature could provide the best
conditions for running the gel. It is important to keep the gels
cold while running, otherwise the lower part of the gels may
become distorted and show wavy bands. Putting the gel appa-
ratus on ice during running could minimize overheating of the
apparatus and distortion of the gel.
8. SDS is a strongly denaturing anionic detergent which unfolds
and fully denatures all proteins including MMPs, essentially
disregarding secondary structures or hydrophobic domains,
and generates SDS-protein complexes that are mostly charac-

terized by a uniform charge-to-mass ratio. This makes SDS-
PAGE in general a very simple and reliable technique for
protein separation and molecular mass determination. The
ratio of 1.4 g of SDS bound per gram of protein is often quoted
as a typical stoichiometric value [67]. When the proteins are
saturated with micelles of SDS (SDS-protein), this amount of
highly charged surfactant molecules is sufficient to overwhelm
the intrinsic charges on the protein chains so that the net
charge per unit of mass becomes almost constant, thus allow-
ing the protein chains to separate through SDS-PAGE, mostly
according to their molecular weight [68].
9. Pro-MMPs are secreted as inactive zymogens with an inhibi-
tory propeptide domain. The pro-MMP architecture in which
Cys73 is located in the vicinity of the Zn2+ ion makes the
Cys73-Zn2+ complex vulnerable to disruption by multiple stim-
uli. Dissociation of the Cys73 residue from the Zn2+ ion
“switches” it from a non-catalytic to catalytic Zn2+. Because
the sequences surrounding Cys73 in the propeptide and the
Zn2+-binding site in the catalytic domain of the MMPs are
highly homologous, the “cysteine switch” mechanism applies
to all MMPs. For example, during the electrophoresis step of
the gelatin zymography experiment, the propeptide is unfolded
in the denaturing conditions induced by SDS. After electro-
phoresis, other non-ionic detergents such as Triton X-100 are
used in the renaturing buffer to replace SDS and remove it
from the SDS-complex. This allows the pro-MMPs in the sam-
ple to renature, become partially refolded, and autoactivate,
resulting in the appearance of a partially catalytically active pro-
MMP portion of the originally inactive pro-MMPs. Only about
35% of the MMP catalytic activity is recovered during the pro-
tein refolding [63], which may not represent the true biologi-
cal activity of the pro-MMP when activated in vivo. Because
these propeptides are covalently anchored to the enzyme pro-
forms, the pro-MMPs are detected at higher molecular weights
than the activated MMPs from which the propeptides are
cleaved off. Furthermore, non-covalently bound complexes,
such as TIMP-MMP complexes, are dissociated by SDS during
the electrophoresis step [56, 64, 69]. Hence, the gelatinolytic
bands of the zymography may not be a measure of the net
MMPs activity present in the sample, but should rather be seen
as a measure of potential activity of MMPs [56, 69, 70].
10. The incubation time of the gel in the developing buffer is criti-
cal for proper renaturation and proteolysis. Since the appear-
ance of the gelatinolytic bands depends on enzymatic activity,
changing the incubation time will affect the intensity of the
bands. Incubating the gel in the developing buffer at 37 °C for
4 h in a closed tray may be sufficient to detect the gelatinolytic

bands. However, in most cases, overnight incubation (16–
18 h) may be needed to obtain better resolution and reproduc-
ible results. If the bands remain barely visible, it may be
necessary to develop the gels for a longer period of time, even
up to 72 h.
11. The identity of the MMP type in the gelatinolytic band is usu-
ally determined by comparing the band location with known
molecular weight standards run simultaneously in the same
gel. This could also help in discerning the latent inactive
from the active forms of MMPs [51]. Also, pro-MMPs are
usually activated in a process involving the generation of an
inactive intermediate forms which are then processed to gen-
erate the fully mature active forms [71, 72]. Some commer-
cially available molecular weight standards contain a reducing
agent, and when they are used under nonreducing condi-
tions, they may indicate different molecular weights [51].
Also, detection of small differences in molecular weight
between intermediate and fully active MMP species would
require further optimization of the conditions to enhance
the sensitivity of the zymography assay. Hence, for an exact
identification of MMPs, Western blot analysis using specific
antibodies should be performed. While Western blot analysis
is more specific than zymography, antibodies may not be
sensitive enough to detect low levels of MMPs. On the other
hand, while zymography is more sensitive than Western blots
in detecting small amounts of MMPs, some of the limita-
tions regarding the resolution of gelatin zymography may
make it difficult to analyze the data. Both gelatin zymogra-
phy and Western blots techniques could complement each
other in studying MMPs.
12. Gelatin zymography can been used to detect the changes in
specific MMP activity in vascular remodeling and angiogen-
esis [1], and in the uterine and vascular changes associated
with pregnancy [8]. We have recently used gelatin zymogra-
phy to measure the changes in MMP activity during uterine
wall stretch and in response to sex hormones during preg-
nancy in rats [54]. We found that oxytocin-induced contrac-
tion of uterine strips was reduced in pregnant compared with
virgin rats. Gelatin zymography showed increased activity of
MMP-2 and -9 in uterus of pregnant versus virgin rats.
Prolonged stretch of uterine strips of virgin rats was associ-
ated with reduced contraction and enhanced activity of
MMP-2 and -9. Treatment of stretched uterus of virgin rats
with 17β-estradiol (E2) or progesterone (P4) or E2 + P4
caused further reduction in contraction and increases in
MMP activity. MMP-2 and -9 decreased oxytocin-induced
contraction in uterus of virgin rat. These data suggested that

during pregnancy uterine stretch and increased sex hormone
levels cause increases in the activity of MMP-2 and -9, which
in turn reduce uterine contraction and enhance uterine
relaxation [54].
13. Gelatin zymography can be used to measure the changes in
MMP activity in vascular disease. We have used gelatin zymog-
raphy to measure the changes in uteroplacental and vascular
MMPs in an animal model of hypertension in pregnancy pro-
duced by reduction in uterine perfusion pressure (RUPP)
[73]. We observed a decrease in gelatinase activity of MMP-2
and -9 in uterus, placenta, and aorta of RUPP compared with
normal pregnant rats. Also, collagen was more abundant in
uterus, placenta, and aorta of RUPP than in those of Norm-
Preg rats. The antiangiogenic factor-soluble fms-like tyrosine
kinase-1 (sFlt-1) decreased MMP activity in uterus, placenta,
and aorta of normal pregnant rats, and vascular endothelial
growth factor (VEGF) reversed the decreases in MMPs in tis-
sues of RUPP rats. These observations suggested that placental
ischemia and antiangiogenic sFlt-1 decrease uterine, placental,
and vascular MMP-2 and -9, leading to increased uteroplacen-
tal and vascular collagen and growth restriction in hypertensive
pregnancy, and that angiogenic factors may reverse the decrease
in MMP activity and enhance uteroplacental and vascular
growth in preeclampsia [73].
14. Gelatin zymography can be used to test the specificity and effi-
cacy of MMPIs. Selective MMPIs can be added to a tissue
sample ex vivo to determine if they reduce MMP activity and
alter tissue function. MMPIs can also be added to the gel (or
part of a gel cut in half) during the incubation in the develop-
ing buffer to determine if they decrease the activity of the
MMP of interest. The optimal dose and reaction time of
MMPIs with MMPs can be determined by performing a
concentration- response curve and time course studies. We
have recently tested the effects of MMPIs on uterine function
and MMP activity. We found that pretreatment of uterine
strips with the MMPIs SB-3CT, BB-94, or Ro-28-2653
(10−6 M) did not change oxytocin contraction in virgin uterus,
but enhanced contraction in uterine strips of pregnant rats.
Also, gelatin zymography revealed that the intensity of the
pro-MMP-2-, MMP-2-, and MMP-9-digested gelatin bands
was reduced in uterine strips treated with the MMP-2/MMP-9
inhibitor IV (SB-3CT, 10−6 M) compared with control non-
treated uterine strips of virgin or pregnant rats [54]. These
observations support the contention that MMPIs could be
used to test the role of changes in MMP activity in modulating
the function of different systems.
15. There is mounting evidence that MMPs regulate tissue

remodeling under physiological and pathological conditions,
and reliable detection and quantitative methods such as
zymography are needed to determine the role of MMPs in
various processes. This classical technique for measuring
MMP activity is a highly sensitive, cost-effective, and rela-
tively simple to perform. Modification of the substrate in the
zymography assay could enhance the detection spectrum to
include MMPs with different substrate preferences.
Improvement in the sensitivity and accuracy of gelatin
zymorgraphy will further enhance its value in assessing the
changes in MMP activity in biological samples and in disease
conditions. Tissue remodeling is controlled by a balance
between endogenous TIMPs and MMPs such that an
increase in TIMP/MMP activity ratio would decrease ECM
protein degradation and vice versa. Reverse zymography
could be utilized to test the activity of endogenous TIMPs
(Fig. 4). Also, synthetic MMPIs have been evaluated as diag-
nostic and therapeutic tools in cancer, autoimmune, and vas-
cular disease [15]. Zymography could be a valuable technique
to test the specificity and efficacy of MMPIs and could help
in the development of more potent and selective inhibitors
for specific MMPs.
Fig. 4 TIMP activity in uterus of pregnant rats. Uterine tissue strips from normal
pregnant rats were homogenized and prepared for reverse zymography.
Separating gel was prepared as in regular gelatin zymography experiment
except that MMP-9 (0.13 μg/mL) was added. TIMP-1 appears as a darker blue
band of the undigested substrate against a faint blue background of the degraded
gelatin substrate
Acknowledgments
This work was supported by grants from National Heart, Lung,

and Blood Institute (HL-65998, HL-98724, HL-111775).
Dr. Zongli Ren was a visiting scholar from the Department of
Cardiovascular Surgery, Renmin Hospital of Wuhan University,
Wuhan 430060, Hubei, PR China, and a recipient of scholarship
from the China Scholarship Council. Dr. Juanjuan Chen was a vis-
iting scholar from the Department of Obstetrics & Gynecology,
The Third Affiliated Hospital of Guangzhou Medical University,
Guangzhou, China 510150.
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Chapter 9
Detection and Characterization of Bacterial Proteinases

Using Zymography
Madathiparambil G. Madanan and Ambili Mechoor
Abstract
Proteinases play a crucial role in invasion and pathogenesis of bacteria, especially the extracellular and
membrane-bound forms. Analysis of these proteinases demands the isolation by retaining the enzymatic
activity. The isolation procedures maintaining the native structure of the enzyme in its soluble form are
also of extreme importance. The qualitative analyses of these proteinases are carried out by electrophoresis
and zymography. Enzymatic characterization based on the effect of inhibitors and activators on gelatinase
activity also can be assessed using this zymography. The membrane-bound proteinases can be isolated in
their native and soluble form, still retaining the activity using 6-aminocaproic acid and sodium deoxycho-
late; the procedure of which is explained in this chapter.
Key words Bacteria, Gelatinase, Proteinase, Zymography, Leptospira, Extracellular matrix
1 Introduction
Degradation and remodeling of the extracellular matrix (ECM)

components play a vital role in maintaining the physiological tissue
functions. Abnormal degradation of the ECM components also
transpires in several pathological conditions like cancer, hepatitis,
and in several bacterial infections as well. One of the major ECM
targets that undergo drastic degradation is collagen which is exe-
cuted primarily by a group of proteinases, which includes collage-
nases and gelatinases [1–3]. Several of these enzymes are produced
by bacteria as well [4]. Bacterial proteinases are mainly seen in two
forms, viz.: the secretory forms and the membrane-bound forms
[5–8]. The usual extraction procedures for the total proteins
including membrane proteins involve the use of strong detergents
and chaotropic ions which can affect the enzymatic activity. Hence,
these procedures implemented for enzyme extraction cannot be
implemented for the isolation of the membrane-bound bacterial
gelatinases and collagenases as it may affect the enzyme activity. An
extraction procedure which preserves the enzyme activity and
103
104 Madathiparambil G. Madanan and Ambili Mechoor
s olubility only can enable the electrophoretic separation and facili-

tate zymography development for detection of proteinase activity.
Described below is an isolation procedure of the total pro-
teins including the membrane-bound bacterial gelatinases using
sodium deoxycholate and 6-aminocaproic acid. The procedure
isolates the enzymes in its intact native soluble form, which
enables electrophoretic separation and zymographic detection.
Sodium deoxycholate, a bile salt-related anionic detergent, can
solubilize the lipid bilayer of the bacterial membrane releasing
the bound proteinases [9–11]. The protein extracted by this
procedure may not be readily soluble. The routine method of
protein solubilization is using sodium chloride, which cannot
be used here as it interferes with electrophoretic separation.
Hence, as an alternate safe solution, 0.75 M 6-aminocaproic
acid is used to solubilize the extracted proteins of outer mem-
brane [12, 13]. The 6-aminocaproic acid is not found to be
interfering with the electrophoresis and can be loaded mixing
with the sample buffer.
1.1 Zymography Zymography is based on the following principles: (1) during elec-
trophoresis, the substrate is retained in the gel; (2) proteinase/gel
atinase/collagenase activity is reversibly inhibited by SDS during
electrophoresis. An additional advantage of zymography is that
both the proenzymes and the active forms of enzymes can be dis-
tinguished on the basis of their molecular weight [14].
Zymography is a powerful technique for analyzing hydro-
lytic enzymes based on substrate degradation. During zymogra-
phy, the proteinases are separated by electrophoresis under
denaturing, nonreducing conditions. The separation occurs in a
polyacrylamide gel containing a specific substrate that is copoly-
merized with the acrylamide [15]. During electrophoresis,
sodium dodecyl sulfate (SDS) denatures the proteinases into an
inactive form. On completion of the electrophoretic run, the gel
is washed with Triton X-100, which causes the exchange of the
SDS with Triton X-100. This helps in partial renaturation and
recovery of the enzyme activity [16]. Subsequently, the gel is
incubated in the appropriate activation buffer. During incuba-
tion, the concentrated, renatured proteinase in the gel will
digest/degrade the impregnated substrate. After incubation, the
gel is stained with Coomassie blue, and the proteinase activity is
detected as clear bands against a blue background of undegraded
substrate. The clear bands in the gel can be quantified by densi-
tometry if needed.
1.2 Bacterial This method is used to find all expressed gelatinases of the labora-
Proteinase Detection tory grown Leptospira—a spirochete pathogen causing
Using Zymography Leptospirosis. The results indicated the presence of 12 gelatinases
in the molecular mass range of 240–32 kDa, representing different
Bacterial Proteinases Zymography 105
Fig. 1 Zymography of total protein extracted from Leptospira using 50 mM

Tris–HCl, pH 7.2, containing 0.5% deoxycholate and 0.75 M 6-aminocaproicacid.
Molecular weight standard (STD), 1 μL (~70 μg protein) Human serum (S), and
50 μg Leptospira protein extract (L), loaded on 10% zymography gel
types of proteinases (Fig. 1). The protein extract can be pretreated

with aminophenyl mercuric acetate (APMA) to bring the proen-
zymes into active form. Treatment with different activators and
inhibitors of proteinases was also carried out in the same buffer
system used for the extraction (Fig. 2). This enabled further char-
acterization of the proteinases avoiding further buffer exchange
and loss of solubility of the membrane-bound forms. Furthermore,
this helped in the direct application of the test onto the zymography
to assess the change in activity.
Fig. 2 Effect of inhibitors on leptospiral proteinase activity: Zymographic analysis on 10% zymography gel.
Leptospira proteinase activity was tested in the presence of EDTA, EGTA, o-PA, or PMSF. The inhibitors were
added reaching the indicated concentration and incubated for 18 h at 37 °C. Each well contains 1 μL (~70 μg
protein) Human serum (S), and 5 μg protein from 46 kDa region of gel filtration fraction of Leptospira total
protein extract (L)
2 Materials
2.1 Chemicals 1. Gelatin from bovine skin, Triton X-100, sodium deoxycholate,
6-aminocaproic acid, 1,10-phenanthroline monohydrate
(o-PA), phenylmethylsulfonyl fluoride (PMSF), ethylenediami-
netetraacetic acid (EDTA), ethylene glycol tetraacetic acid
(EGTA), deionized water to attain a sensitivity of 18 MΩ cm

at 25 °C.
2. All other reagents/chemicals are of analytical grade (see Note 1).
2.2 Instruments 1. Electrophoresis apparatus: Any brand/model that can cast

10 × 7 cm2 or more sized gel of thickness 1 mm.
2. Gel trays: Gel trays must be of at least 5% larger in dimensions
and having smooth and flat surface with lid.
3. Incubator: For incubating the gel trays for 18 h at 37 °C.
4. Shaker: Rocking or rotary shaker with low speed for staining
the gel.
2.3 Buffers 1. Acrylamide-bis acrylamide solution (30% T, 3% C): Dissolve

and Stock Solutions acrylamide 29.1 g and bis-acrylamide 0.9 g in 40 mL water.
Make up to 100 mL with water and filter through a Whatman
2.3.1 Stock Solutions
grade 1 filter paper. Store the stock solution at 4 °C in an
for Electrophoresis
amber-colored bottle.
2. Resolving gel buffer (1.5 M Tris–HCl, pH 8.8): Dissolve
18.18 g Tris base with 80 mL water. Adjust the pH to 8.8 by
titrating with HCl. Make up the volume to 100 mL with water
and store at 4 °C.
3. Stacking gel buffer (0.5 M Tris–HCl, pH 6.8): Dissolve 6.06 g
Tris base with 80 mL water. Adjust the pH by titrating with
HCl to 6.8. Make up the volume to 100 mL with water and
store at 4 °C.
4. Sodium dodecyl sulfate 10%: Dissolve 10 g SDS in 80 mL of
water and make up the volume to 100 mL and store at room
temperature.
5. Ammonium persulfate (APS) 10%: Dissolve 100 mg ammo-
nium persulfate in 1 mL of water and store at 4 °C. This stock
is stable up to 2 weeks.
6. Gelatin 10%: Dissolve 100 mg gelatin in 1 mL of water by
warming at 40 °C. This can be stored in aliquots of 100 μL at
−20 °C. Thaw to dissolve completely before use. This stock is
stable up to 2 weeks.
7. 10× Electrode buffer (0.025 M Tris–HCl, 0.192 M glycine,
0.1% SDS pH 8.3): Dissolve 30.28 g Tris base, 144 g glycine,
and 10 g SDS in 800 mL of water. Make up the solution to
1000 mL. The pH of the solution thus prepared would be
approximately 8.3. The buffer required for running the
electrophoresis may be diluted to 1× and the stock buffer

stored at 4 °C.
8. Sample Buffer (2×): Take 5 mL of water and 2.5 mL of 0.5 M
Tris–Cl, pH 6.8 (Resolving Gel Buffer as above) in a 10 mL grad-
uated tube and dissolve 200 mg SDS slowly without clogging and
foaming. Add 2 mL of glycerol mix completely and dissolve 20 μg

of bromophenol blue. Make up the volume to 10 mL and store at
room temperature.
2.3.2 Reagents 1. Wash Buffer: Phosphate buffered saline containing 5 mM

for Protein Extraction MgCl2: Take 100 mL of PBS and dissolve 47.6 mg of MgCl2.
2. Extraction buffer (50 mM Tris–HCl, pH 7.2, containing 0.5%
deoxycholate and 0.75 M 6-aminocaproicacid): 500 mM Tris–
Cl pH 7.2 was prepared by dissolving 6.06 g Tris base in
80 mL of water and pH adjusted to 7.2 with HCl. The volume
made up to 100 mL with water and stored at 4 °C.
Take 1 mL of 500 mM Tris–HCl pH 7.2 in a 10 mL graduated
tube and add 5 mL of water. Dissolve 50 mg of deoxycholate
and 984 mg of 6-aminocaproicacid (see Note 2). Make up the
volume to 10 mL while dissolving the reagents completely.
Store at 4 °C (see Note 3).
2.3.3 Reagents 1. Renaturing Buffer (2.5% Triton X-100 solution): Dissolve

for Zymography 2.5 mL of Triton X-100 in 97.5 mL of water (see Note 4).
2. Stock 500 mM Tris–Cl: 500 mM Tris–Cl pH 7.5 is prepared
by dissolving 6.06 g Tris base in 80 mL of water and pH
brought to 7.5 with HCl. Volume made up to 100 mL with
water and store at 4 °C.
3. Stock 250 mM CaCl2: Dissolve 2.77 g CaCl2 in 100 mL water
and store at room temperature.
4. Activation buffer (50 mM Tris–HCl pH 7.5, 1% Triton X-100
and 25 mM CaCl2): Dissolve 1 mL of Triton X-100 in 79 mL
of water. Add 10 mL of 500 mM Tris–Cl pH 7.5 and 10 mL
of 250 mM CaCl2 (see Note 5).
5. Stock 500 mM EDTA: Dissolve 18.61 g EDTA in 80 mL of
water, add NaOH pellets (approximately 2 g) to bring the
pH 8, adjust the volume to 100 mL.
6. Stock 500 mM EGTA: Dissolve 19.02 g EGTA in 80 mL of
water, add NaOH pellets (approximately 6.5 g) to bring the
pH 8, adjust the volume to 100 mL.
7. Stock 50 mM o-PA: Dissolve 99.11 mg o-PA in 10 mL of
ethanol.
8. Stock 100 mM PMSF: Dissolve 17.14 mg PMSF in 10 mL of
isopropanol and store at −20 °C.
2.3.4 Reagents 1. Coomassie blue stain: To make 100 mL Coomassie blue stain
for Staining mix 40 mL methanol (or ethanol), 10 mL acetic acid, 50 mL
of water, and dissolve 10 mg Coomassie brilliant blue R 250.
2. Destaining solution: Destaining solution is a mixture of 40 mL
methanol (or ethanol), 10 mL acetic acid, and 50 mL water
(total volume will be 100 mL).
3 Methods
3.1 Electrophoresis 1. Assembly of gel cassette: The glass plates should be thoroughly
cleaned using detergent without leaving any greasy areas left
on the surface. Fingerprints on the glass surface also should be
avoided. Align the plates perfectly without any leak. The leak
can be checked by pouring water into the casting setup and
wait for few minutes to find the leak based on decrease in the
water level. The water should be then drained and the inner
side of the casting setup should be thoroughly cleaned with
long strips of filter paper.
2. Resolving gel: The upper level of resolving gel may be decided
by placing the comb in position and marking the bottom of each
well using a marking pen. Make the resolving gel mix by adding
the components at required volume, with reference to the
Table 1, to cast the gel. The system mentioned (8 × 10 cm2)
required 10 mL of resolving gel mix. Add ammonium persulfate
just before casting. Pour the resolving gel mix into the casting
chamber through the middle portion of the setup to about
5 mm below the marking of bottom line of the wells. Care
should be taken to avoid air bubbles. Slowly layer some water
about 1 mm level on the top of the gel to make an even layer and
removing bubbles. Allow to set the gel for 1 h (see Note 6).
3. Stacking gel: Decant the water from the top of the gel by tilt-
ing the gel setup to one side so that the water gets collected to
the corner which can be removed using a narrow strip of filter
paper. Make the stacking gel mix by adding the components at
required volume, with reference to the Table 2, to cast the gel.
Pour the staking gel onto the resolving gel and place the comb
in position to form the wells with about 5 mm stalking gel
between the resolving gel and the bottom of wells. Wait for at
least 3 h for complete polymerization. This is required for
Table 1
Composition of resolving gel
Resolving gel 10% T 10 mL 20 mL 50 mL

H2O 3.86 7.71 19.27
Tris–HCl pH 8.8 2.5 6.68 16.7
ABA 30% 3.34 5 12.5
10% Gelatin 0.1 0.2 0.5
10% SDS 0.1 0.2 0.5
TEMED 0.006 0.012 0.03
10% APS 0.1 0.2 0.5
Table 2
Composition of stacking gel
Stacking gel 3.5% T 3 mL 6 mL 10 mL

H2O 1.84 3.68 6.13
ABA 30% 0.35 0.7 1.17
Tris–HCl pH 6.6 0.75 1.5 2.5
10% SDS 0.03 0.06 0.1
TEMED 0.006 0.009 0.012
10% APS 0.03 0.06 0.1
good cross-linking of the gelatin. Remove the lower spacer and

fix the gel on the electrophoresis apparatus. Slowly keep the
gels in the tank containing 1× electrode buffer without having
trapped air in the bottom of the gel.
4. Preparation of Sample: Bacterial culture (10 mL culture of
Leptospira) is centrifuged at 5000 × g for 10 min and the pellet
shall be washed two times with 1 mL of wash buffer and cen-
trifuged at 5000 × g for 10 min at room temperature. To the
pellet obtained after second wash, add 100 μL of extraction
buffer and vortex for 5 min. The supernatant of the extract will
be centrifuged 12,000 × g for 10 min at room temperature and
the supernatant can be used as the sample. Required amount
(about 50–100 μg protein per well) of this sample should be
mixed with equal amount of 2× sample buffer and incubate at
37 °C for 30 min before loading onto the gel.
5. Loading the sample: Load the sample into the wells carefully
with a micropipette and gel loading tips to avoid cross-
contamination of samples. Carefully top-up the remaining por-
tion of the well and the entire top of the gel with electrode
buffer without disturbing the loaded samples. Fill the upper
tank of the electrophoresis unit also using the electrode buffer.
6. Running the gel: Electrophoresis must be carried out at 4 °C
in a cold room. Alternately, electrophoresis can be performed
inside a refrigerator (see Note 7). Electrophoresis unit is
switched on initially at 6 mA per gel until the dye front enters
the resolving gel. Increase the current to 12 mA per gel for
remaining run until the dye front reaches 1 cm above from the
bottom level of the gel (see Note 8). In a mini gel format
(8 × 10 cm), the entire run takes about 2 h.
7. Marking of bands of the standard: On completing the electropho-
resis, carefully open the glass plates using a plastic spatula or won-
der wedge-plate separation tool and mark the position of dye front
and prestained marker by punching holes or scanning the gel.
3.2 Zymography 1. Place the gel submerged in renaturing solution in a gel tray.
Incubate at room temperature for 30 min without shaking
(see Note 9).
2. Remove the renaturing solution and add activation buffer in
order to completely submerge the gel.
3. Incubate in an incubator at 37 °C for 18 h.
3.3 Characterization For protein characterization, the gel will be subjected to incuba-
of Proteinases tion with different reagents and concentrations to be tested. To
achieve this, the reagent to be tested must be added to the activa-
tion buffer. This can be made from stock solution of the reagents
which is made in appropriate solvents. This stock must be added,
in order to reach the required final concentration of the reagent,
while making the activation buffer by replacing equivalent amount
of water from the buffer composition.
1. After renaturation the gel should be kept on a clean glass plate.
2. Lanes to be tested in different reagents must be cut through
the inter-lane space recognizable through the space between
the wells on top and space between the color of dye front at
the bottom.
3. Mark the orientation of gel on each lane.
4. Place the gel in a small tray sufficient to occupy the gel. Small
trays used for incubation of Western blots or 15 mL screw-
caped centrifuge tubes (for single lane from 8 × 10 cm gel and
careful to avoid folding and partial immersion in the buffer)
may be used. For each lane from 8 × 10 cm2 format gel 10 mL
of the activation buffer will be sufficient.
5. Incubate the gels in an incubator at 37 °C for 18 h for activity
(see Note 10).
3.4 Staining 1. After the incubation, remove the activation buffer and briefly
rinse the gel with water for 1 min. This removes the extra
Triton X-100 on the gel which may cause unnecessary foaming
while shaking.
2. Add staining solution into the tray submerging the gel
completely.
3. Keep the gel shaking at lower speed to avoid damage to the gel.
4. Staining may be continued about 1 h or until the gel becomes
completely blue.
5. Remove the stain and rinse the gel for 1 min with water to
remove extra stain from the surface of the gel.
6. Add destaining solution into the tray and keep shaking.
7. Change the destaining solution several times until the desired
band contrast is observed with respect to the blue background.
8. Scan the gel using a gel documentation system or document

scanner.
9. Determine the molecular mass of protein bands with respect to
the protein standards.
4 Notes
1. The reagents can be stored at room temperature (unless indi-

cated otherwise). The waste disposal regulations should be
strictly followed while disposing waste materials.
2. Crystallization of the reagents may occur at 4 °C which can be
redissolved at room temperature before extraction procedure
3. The 6-aminocaproic acid is also known as EACA and is
reported to inhibit chymotrypsin, Factor VIIa, lysine carboxy-
peptidase, plasmin, and plasminogen activator [17–21]; but
the inhibitory activity was not tested in our experiment, as the
samples were of bacterial origin.
4. This is done by taking Triton X-100 with a wide bore pipette
and pouring into water while continuously stirring on a mag-
netic stirrer.
5. The volume required may vary in order to completely sub-
merge the gel in the buffer with respect to the size of the gel
tray used.
6. We found that the 1 mm thick gel is ideal for good resolution
and contrast of bands. The thinner gels make lighter back-
ground and the thicker ones make darker background with less
visibility of bands. This may be due to more destaining and less
amount of gelatin in the thinner gels in contrast to high
amount of gelatin and difficulty in destaining in the thicker
gels. Also, the gelatin in the thicker gels may not have com-
pletely digested by the proteinase due to requirement of more
time; the enzyme may get degraded during the course of time
or may not be having proper renaturation and buffering due to
thickness of the gel.
7. In our electrophoresis system, about three fourth lower por-
tions of the plates are submerged in the electrode buffer
(anode) tank. This system exchanges heat generated during
electrophoresis and prevents loss of proteinase activity. We
have used prechilled electrode buffer (stored at 4 °C) which
had an advantage of avoiding requirement of cold room or
refrigerator for running the gel.
8. In case of other gels the voltage and run time can be calculated
as per the standard SDS–PAGE.
9. In many publications and according to other procedures, shak-

ing enhances SDS elimination, as well as multiple washes
(3 × 15 min each wash) are suggested. Sometimes with shaking
conditions we have noticed that the background become pale
and bands not sharp. We assume that this is because the gel is
not fixed and, while incubation, the protein leaches out and
the bands spread more. Even without shaking, we were able to
get good bands.
10. In the experiment we have tested the inhibition of proteinases
with different inhibitors such as EDTA (0, 50 and 100 mM),
EGTA (0, 50 and 100 mM), o-PA (0, 2 and 16 mM), PMSF
(0, 2 and 16 mM) by developing zymogram gels in activation
buffer containing these reagents incubated for 18 h at 37 °C
as shown in Table 3. The results show differences in band
intensity caused by the presence of the inhibitors (Fig. 2).
While EDTA, EGTA, and o-PA inhibit metalloproteases by
chelation, PMSF inhibits serine or cysteine proteases. Similarly,
activation by different metal ions, effect of pH, and tempera-
ture on the proteinase activity can be demonstrated [4, 7] in
order to characterize the proteinases.
Table 3
Modification of activation buffer with inhibitors
Final concentration EDTA EGTA o-PA PMSF

(mM) of inhibitors
in activation buffer 0 50 100 0 50 100 0 2 16 0 2 16
Water (mL) 7.9 6.9 5.9 7.9 6.9 5.9 7.9 7.5 4.7 7.9 7.7 6.3
500 mM EDTA 0 1 2 – – – – – – – – –
(mL)
500 mM EGTA – – – 0 1 2 – – – – – –
(mL)
50 mM o-PA (mL) – – – – – – 0 0.4 3.2 – – –
100 mM PMSF – – – – – – – – – 0 0.2 1.6
(mL)
500 mM Tris-HCl, 1 1 1 1 1 1 1 1 1 1 1 1
pH 7.5 (mL)
250 mM CaCl2 1 1 1 1 1 1 1 1 1 1 1 1
(mL)
Triton X-100 (mL) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Total volume (mL) 10 10 10 10 10 10 10 10 10 10 10 10
Acknowledgments
All the demonstrated experiments were carried out by Mr.

Sandhanakrishnan Cattavarayane and the work was supported by
grant Nos. SR/SO/HS-29/2005 and SR/S0/HS/0108/2012,
SERB Division, Department of Science and Technology, New
Delhi, India to M.G.M.
References
1. Ambili M, Jayasree K, Sudhakaran PR (1998) 11. Seddon AM, Curnow P, Booth PJ (2004)
60K gelatinase involved in mammary gland Membrane proteins, lipids and detergents: not
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Biochim Biophys Acta 1403:219–231 1666:105–117
2. Birkedal-Hansen H, Moore WG, Bodden MK 12. Schagger H, Cramer WA, Von Jagow G (1994)
et al (1993) Matrix metalloproteinases: a Analysis of molecular masses and oligomeric
review. Crit Rev Oral Biol Med 4:197–250 states of protein complexes by blue native elec-
3. Sato H, Takino T, Okada Y et al (1994) A trophoresis and isolation of membrane protein
matrix metalloproteinase expressed on the complexes by two-dimensional native electro-
surface of invasive tumour cells. Nature 370: phoresis. Anal Biochem 217:220–230
61–65 13. Schagger H, Von Jagow G (1991) Blue native
4. Madathiparambil MG, Cattavarayane S, electrophoresis for isolation of membrane pro-
Manickam GD et al (2011) A zymography tein complexes in enzymatically active form.
analysis of proteinase activity present in Anal Biochem 199:223–231
Leptospira. Curr Microbiol 62:917–922 14. Woessner JF Jr (1995) Quantification of matrix
5. Haiko J, Suomalainen M, Ojala T et al (2009) metalloproteinases in tissue samples. Methods
Invited review: breaking barriers—attack on Enzymol 248:510–528
innate immune defences by omptin surface 15. Leber TM, Balkwill FR (1997) Zymography: a
proteases of enterobacterial pathogens. Innate single-step staining method for quantitation of
Immun 15:67–80 proteolytic activity on substrate gels. Anal
6. Le Sage V, Zhu L, Lepage C et al (2009) An Biochem 249:24–28
outer membrane protease of the omptin family 16. Snoek-Van Beurden PA, Von Den Hoff JW
prevents activation of the Citrobacter roden- (2005) Zymographic techniques for the analy-
tium PhoPQ two-component system by anti- sis of matrix metalloproteinases and their
microbial peptides. Mol Microbiol 74:98–111 inhibitors. Biotechniques 38:73–83
7. Madathiparambil MG, Cattavarayane S, 17. Adelman B, Rizk A, Hanners E (1988)
Perumana SR et al (2011) Presence of 46 kDa Plasminogen interactions with platelets in
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8. Sole M, Scheibner F, Hoffmeister AK et al Catheptic carboxypeptidase B as a major com-
(2015) Xanthomonas campestris pv. vesicatoria ponent in “T-cell activating factor” of macro-
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10. Lichtenberg D, Ahyayauch H, Alonso A et al 21. Soter NA, Austen KF, Gigli I (1975) Inhibition
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Sci 38:85–93 tem. J Immunol 114:928–932
Chapter 10
A Sensitive, Rapid, and Specific Technique

for the Detection of Collagenase Using Zymography
Shivcharan Prasad and Ipsita Roy
Abstract
In-gel zymography is a commonly employed tool to identify active enzymes in a quantitative and qualita-
tive manner. In this work, apart from the incorporation of substrate which is traditionally employed in
zymography, the identification of collagenase by incubation of the enzyme resolved on a polyacrylamide
gel with substrate solution is described. The two methods are quite fast and result in specific detection of
bacterial collagenase.
Key words Collagen, Collagenase, Matrix metalloproteinase, Zymography
1 Introduction
Matrix metalloproteinases (MMPs) are a group of structurally and

functionally related calcium-dependent endopeptidases which act on
cellular matrices. These are synthesized as inactive precursors and are
converted into the functionally active form as per physiological need.
An imbalance in the ratio of MMPs and their inhibitors is a charac-
teristic of many disease conditions like cancer metastasis, cardiovas-
cular diseases, rheumatoid arthritis, etc. [1]. Hence, accurate
measurement of active MMPs is important. Different methods have
been reported in the literature to assay the activity of matrix metal-
loproteinases [2]. Although native proteins have been employed as
substrates, most of the enzymatic assays for matrix metalloprotein-
ases rely on synthetic substrates as the natural substrates are difficult
to dissolve in aqueous buffers. These reactions are also quite slow.
The synthetic substrates are often labeled. Triple helical peptides
derived from collagen have also been employed [2]. Immunological
methods like ELISA have limited application as they are unable to
distinguish between the inactive precursor, isoforms, the degraded
protein, and the active enzyme. Zymography (visualization of an
enzyme on a gel by substrate conversion) is a commonly used tool
115
116 Shivcharan Prasad and Ipsita Roy
for measuring the activity of MMPs, mainly gelatinases and collage-

nases [3–6]. In fact, the initial identification of an active collagenase
involved in collagen degradation employed zymography [7].
Bacterial collagenases are defined by their ability to cleave helical
structures in fibrillar collagen in a physiological milieu [8]. The col-
lagenase ColG from Clostridium histolyticum is classified as a Class I
collagenase [9] while ColH from the same organism is placed in
Class II [10], based on the presence and arrangement of their
domains and subdomains. Bacterial collagenases are employed in a
wide range of applications including debridement of wounds and
burns and other areas of medical interest, food technology, tannery,
pharmaceutical and cosmetic industries [11]. In this work, we have
described two protocols for specific detection of very low amounts
(nanogram level) of collagenases [5]. In the first case, collagen is
incorporated into the polyacrylamide gel matrix while in the second
method, the polyacrylamide gel-immobilized collagenase is incu-
bated with the substrate solution post electrophoretic run. Although
our method is developed using purified collagenase, we show its
applicability in a crude sample containing a mixture of enzymes
including collagenase.
2 Materials
All solutions were prepared in ultrapure deionized water of

18 MΩ cm at 25 °C.
2.1 Material The following materials are used:

Description
1. Crude collagenase containing collagenase Type I (125 U/mg).
2. Collagenase (EC 3.4.24.3, Type VII from Clostridium
histolyticum).
3. Collagen (Type I).
2.2 Preparation 1. Add collagen to 0.013 M HCl (4 mg/mL). Incubate at 60 °C

of Collagen Solution overnight [3].
2. Filter the collagen solution with Whatman grade 1 filter paper
(see Note 1).
3. Adjust the pH of collagen solution to assay condition (see Note 2).
For this, add one-fourth volume of a buffer concentrate prepared
by mixing equal volumes of 0.2 M Na2HPO4 and 0.5 M NaCl.
Adjust pH to 7.4 with 0.1 M NaOH [3]. The final concentration
of collagen is adjusted to 3.2 mg/mL.
2.3 Non-denaturing 1. Prepare 30% (w/v) acrylamide-bisacrylamide solution (30% T,

Polyacrylamide Gel 2.6% C) (29.2:0.8 acrylamide:bisacrylamide). For this, take
Electrophoresis 40 mL distilled water in an amber bottle of 100 mL capacity.
Weigh 29.2 g of acrylamide crystal and 0.8 g N,N′-methylene
Collagenase Zymography 117
bisacrylamide and transfer to the bottle. Stir on a magnetic

stirrer for 30 min (see Note 3). Make up the volume to 100 mL
with distilled water. Filter through Whatman grade 1 filter
paper. Store the solution at 4 °C till use.
2. For 4× resolving gel buffer, weigh 18.15 g of Tris and dissolve
in 80 mL of distilled water in a glass beaker. Adjust the pH to
8.8 with 3 M HCl and make up the final volume to
100 mL. Store the solution at 4 °C till use.
3. For 4× stacking gel buffer, weigh 6.0 g of Tris and dissolve in
80 mL of distilled water in a glass beaker. Adjust the pH to 6.8
with 3 M HCl and make the final volume up to 100 mL. Store
the solution at 4 °C till use.
4. Prepare 10% (w/v) ammonium persulfate (APS) in distilled
water (see Note 4).
5. Prepare electrophoresis running buffer by stirring 6.05 g Tris
buffer and 28.80 g glycine in 900 mL distilled water in a 1 L
graduated cylinder for 30 min. Adjust the pH to 8.3 with 3 M
HCl and make up the final volume to 1 L with distilled water.
6. Prepare sample loading buffer by mixing 100 mM Tris–HCl,
pH 6.8, 0.2% (w/v) bromophenol blue and 20% (v/v)
glycerol.
7. Prepare staining solution by dissolving 250 mg Coomassie
Brilliant Blue R-250 in a solution of 45 mL each of methanol
and distilled water and 10 mL glacial acetic acid.
8. Prepare destaining solution by mixing 375 mL methanol
(37.5% v/v) and 100 mL glacial acetic acid (10% v/v) and
making up the volume to 1 L with distilled water.
3 Method
3.1 Zymography 1. Thaw all solutions at room temperature for at least 15 min.
of Collagenase 2. Prepare the resolving gel by adding 3.2 mL distilled water,
(Protocol 1) 2 mL resolving gel buffer, 2.64 mL acrylamide-bisacrylamide
solution and 963 μL collagen in a 15 mL falcon tube.
3. Add 80 μL ammonium persulfate solution and 6 μL TEMED
to the above. Mix gently and add the solution in-between two
glass plates with the help of a pipette. Stand the gel on a hori-
zontal table for 30–45 min for complete polymerization.
4. Add 1:1 isopropanol solution on the top of the gel.
5. Once the gel has polymerized, decant off the solvent and wash
the top of the gel once with distilled water.
6. Prepare the stacking gel by adding 1.83 mL distilled water,
0.75 mL stacking gel buffer and 0.39 mL acrylamide-bisacrylamide
solution in a 15 mL falcon tube. Add 15 μL ammonium persul-

fate solution and 3 μL TEMED to this solution.
7. Mix gently and add the solution on top of the resolving gel in-
between two glass plates with the help of a pipette.
8. Insert the comb gently between the gel plates and avoid trap-
ping of air bubble below the comb. Allow the gel to stand for
at least 30 min to polymerize.
9. Remove the combs and wash the wells with electrophoresis
running buffer.
10. Mix the collagenase samples with loading buffer (1:1, v/v) and
load in the wells.
11. Fill the upper and lower chambers (mini VE vertical electro-
phoresis system, GE Healthcare) with electrophoresis running
buffer.
12. Apply a constant voltage (150 V, 25 mA) through an external
power supply.
13. After the completion of the run, soak the polyacrylamide gel in
0.01 M sodium phosphate buffer, pH 7.4 containing 0.001 M
Ca2+ for 4 h (see Note 5) (Fig. 1).
14. Stain the gel with staining solution for about 1 h (see Note 6).
15. Destain the gel with destaining solution (see Note 6).
16. Scan the gel on any image scanner with an illuminator.
Fig. 1 Zymography of collagenase by protocol 1. (a) After the completion of the

electrophoretic run, the polyacrylamide gel was soaked in 0.01 M sodium phos-
phate buffer, pH 7.4 containing 0.001 M Ca2+ for 3 h. Lane 1: 2 U collagenase,
Lane 2: 4 U collagenase. The specificity of the method was checked by running
4 μg of BSA (Lane 3) as the control. (b) Electrophoresis of collagenase (Lane 1:
32 mU, Lane 2: 40 mU, Lane 3: 50 mU) was carried out at 4 °C, as described
before. After the completion of the run, the polyacrylamide gel was incubated in
0.01 M sodium phosphate buffer, pH 7.4 containing 0.001 M Ca2+ for 4 h. One
unit of collagenase activity is defined as the amount of enzyme required to pro-
duce 1 μmol of leucine in 5 h, as determined by the ninhydrin method [12], under
standard assay conditions [13]. Reproduced with permission from [5].
3.2 Zymography 1. Prepare 10% crosslinked polyacrylamide gel as described before

of Collagenase (see Subheading 3.1, steps 1–4) but without adding collagen.
(Protocol 2) Insert a comb gently between the gel plates and avoid trapping
of air bubble below the comb.
2. Load collagenase samples in the wells (along with the sample
loading buffer) by mixing equal volume of each (see Note 7)
(Fig. 2).
3. Run the gel at room temperature under constant voltage con-
dition (150 V, 25 mA) in a discontinuous buffer system [14].
4. After the completion of the run, wash the gel twice with dis-
tilled water and soak in a solution containing 20 mL colla-
gen solution, 8 mL 0.05 M sodium phosphate buffer,
pH 7.4 (final concentration 0.01 M) and 28 μL 1 M Ca2+
(final concentration 0.001 M).
5. Repeat steps 14–16 from Subheading 3.1 (see Note 6).
Fig. 2 Zymography of collagenase by protocol 2. Electrophoresis of collagenase

(Lane 1: 1.2 mU, Lane 2: 0.6 mU, Lane 3: 0.2 mU, Lane 4: 0.18 mU, Lane 5:
0.12 mU, Lane 6: 0.03 mU, Lane 7: 0.024 mU, Lane 8: 0.018 mU, Lane 9:
0.012 mU, Lane 10: 0.003 mU) was carried out at 4 °C. (b) Densitometric analy-
sis of the bands was carried out using the Image QuantTL software (GE
Healthcare). Values shown are mean of three independent zymograms. (c)
Electrophoresis of a crude extract of Clostridium histolyticum, known to contain
elastase, clostripain and sulfhydryl protease (Worthington website), apart from
collagenase, was carried out at 4 °C (Lane 1: 7.5 μg protein, Lane 2: 15 μg, Lane
3: 22.5 μg, Lane 4: 30 μg, Lane 5: 37.5 μg, Lane 6: 45 μg, Lane 7: 56.2 μg, Lane
8: 75 μg). Reproduced with permission from [5]
4 Notes
1. Filtration is required to remove undissolved collagen, so that a

homogenous solution can be prepared.
2. The optimal pH for collagenase activity is 7.4.
3. Wear face mask and gloves when weighing and handling acryl-
amide crystals. Residual unpolymerized acrylamide and
bisacrylamide are neurotoxic.
4. It is better to prepare fresh solution every time and store on
ice. Store APS salt in a desiccator at 4 °C.
5. Ca2+ is required for collagenase activity.
6. Both overstaining and overdestaining lead to loss of sensitivity
(Fig. 3). Combination of staining and destaining protocols is
reported to increase sensitivity [15].
7. Samples should not be boiled before loading in the wells.
Fig. 3 Schematic representation of the consequences of overstaining or overdestaining of the zymogram

Acknowledgements
Partial financial support from Department of Science and

Technology is acknowledged. The authors thank K. Thanzami for
her contribution to the work described here.
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Part III
Reverse Zymography and In Situ Zymography

Chapter 11
Reverse Zymography: Overview and Pitfalls

Kanika Sharma and Debasish Bhattacharyya
Abstract
Reverse zymography is a technique by which protease inhibitor(s) in a sample could be electrophoretically
separated in a substrate-impregnated acrylamide gel and their relative abundance could be semi-quantified.
The gel after electrophoresis is incubated with a protease when the impregnated substrate and all other
proteins of the sample are degraded into small peptides except the inhibitor(s) that show clear bands
against a white background. Since reverse zymography cannot distinguish between a protease inhibitor
and a protein that is resistant against proteolysis, the results should be confirmed from inhibition of pro-
tease activity by solution state assay.
Key words Substrate electrophoresis, Inhibitor, Protease digestion, Protease-resistant proteins
1 Introduction
Proteolytic enzymes form the core of human pathophysiology by

executing innumerable degradative as well as developmental func-
tions [1]. Regulation of proteolytic activity by their specific or
nonspecific inhibitors helps in maintaining a pivotal balance
between the synthesis and turnover of proteins [2]. Matrix metal-
loproteinases (MMPs) are calcium-dependent zinc-containing
endopeptidases belonging to metzincin superfamily. Apart from
Extra Cellular Matrix (ECM) degradation, they are involved in cel-
lular proliferation, migration, apoptosis etc. Tissue inhibitors of
metalloproteinases (TIMPs) are the family of four inhibitors:
TIMP-1, TIMP-2, TIMP-3, and TIMP-4. They regulate MMP
activity by chelating the zinc group present at the active site.
Maintenance of balance between MMPs and TIMPs is crucial to
avoid chronic pathological conditions [3]. Several methods have
been developed to analyze MMP/TIMP activity in biological sam-
ples. Zymography and its modification, reverse zymography, are
two such widely used methods.
Zymography as a substrate electrophoretic technique is widely
used to identify proteolytic activity under non-reducing conditions.
125
126 Kanika Sharma and Debasish Bhattacharyya
Post electrophoresis, replacement of SDS by a non-ionic detergent

of low critical micelle concentration (CMC) allows the proteases to
refold partially resulting in a catalytically active enzyme. It is the
method of choice to detect the activity of different matrix metal-
loproteinase (MMP) isoenzymes from a wide range of biological
samples [4]. It also discriminates between functional monomers,
multimers, complexes and degraded products, thus providing an
activity-based evaluation of the biological sample [5, 6].
Reverse zymography is a modified zymographic technique
which enables the detection of protease inhibitors in biological
samples. It facilitates the characterization of inhibitors, their rela-
tive abundance and stability against the protease. This method was
initially developed to identify TIMPs in biological extracts by
copolymerizing gelatin, MMP-2/MMP-9 with acrylamide
whereby TIMP forms a 1:1 complex with MMP which appears as
bands against white background [7]. Figure 1 shows the visual dif-
ference between zymography (white band against blue back-
ground) and reverse zymography (blue bands against white
background).
Basic protocol of reverse zymography involves electrophoretic
separation of sample on a substrate-copolymerized SDS-PAGE.
Post run, the gel is washed with Triton-X 100 to reduce SDS pres-
ent in the gel. This is followed by incubation of the gel in develop-
ing buffer incorporated with specific protease, against which
inhibitor is being searched, for 12–24 h at 37 °C. The protease
digests the background substrate and results in appearance of
bands where the inhibitor is present. Staining and destaining pro-
vides blue bands of protease inhibitor(s) against white background.
Fig. 1 (1) Gelatin zymography of leaf bromelain. (2) Reverse zymography of

Soyabean Trypsin Inhibitors (STI) (25 μg) digested with trypsin (0.5 mg)
Reverse Zymography 127
On the basis of the protease being used, one can deduce informa-
tion regarding the protease family to which the inhibitor belongs.
Similar to zymography, reverse zymography offers advantages like
detection of inhibitors in impure samples, non-interference of
optically absorbing molecules, electrophoretic separation of inhibi-
tors, their relative abundances and molecular weights.
2 Materials
All reagents were prepared using ultrapure distilled water and

stored at 4 °C until otherwise stated.
1. Resolving gel buffer: 3 M Tris–HCl, pH 8.8. Weigh 363.1 g of
Tris and dissolve in approximately 250 mL distilled water.
Adjust the pH with 6 M HCl and complete to 1 L. Store at
4 °C (see Note 1).
2. Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8. Weigh 60.5 g of
Tris and prepare 1 L buffer solution as described in step 1.
Store at 4 °C.
3. 30% acrylamide/bisacrylamide solution: weigh 29.2 g of acryl-
amide (monomer) and 0.8 g of bisacrylamide (cross-linker)
and transfer to 100 mL graduated cylinder containing 50 mL
water. Mix the solution for approximately 30 min on magnetic
stirrer to get a clear solution. Make up the volume to 100 mL
using water, centrifuge and pass through a 0.45 μm filter. Store
at 4 °C in an amber-colored glass bottle covered with alumi-
num foil to protect from light (see Note 2).
4. Ammonium persulfate: 10% solution in water. Always use a
freshly prepared solution.
5. N,N,N′,N′-Tetramethyl-ethylenediamine (TEMED): use as
purchased. Store at 4 °C.
6. Zymography running buffer: 0.025 M Tris–HCl, pH 8.3,
0.192 M glycine, 0.1% SDS. Store at room temperature.
7. Developing buffer: 10 mM Tris–HCl, pH 7.6, 200 mM NaCl,
10 mM CaCl2, 0.02% Brij-35 and variable types of protease
against which the inhibitor is searched for.
8. Staining solution: dissolve 0.025 g of Coomassie Brilliant Blue
in 40 mL methanol, 10 mL acetic acid and make up the solu-
tion to 100 mL.
9. Destaining solution: methanol:acetic acid:water (40:10:50)
(AR/GR grade).
10. Native sample loading buffer (2×): 0.5 mL of 0.5 M Tris-HCl,
pH 6.8, 0.002% bromophenol blue, 0.22 mL glycerol added
to 1.28 mL water.
3 Methods
1. Electrophoresis is performed using a vertical electrophoresis

apparatus with a 10 × 8 × 0.2 cm3 gel cassette.
2. Prepare the 10% resolving gel (% with respect to acrylamide
concentration) by mixing 0.1% substrate (BSA, gelatin, col-
lagen etc as par choice) with 3.32 mL of 30% acrylamide
solution, 1.25 mL of resolving buffer, 0.1 mL 10% SDS,
5.21 mL distilled water, 0.02 mL of TEMED, and 0.1 mL of
10% ammonium persulfate solution. Fill the gel cassette cov-
ering 3/4th of the available space. Carefully cover the resolv-
ing gel layer with water so that the gel surface remains
horizontal. Follow polymerization of the gel by tilting the
cassette. Once polymerized, the water layer is removed by
soaking with a tissue paper (see Notes 3 and 4).
3. Prepare the stacking gel by mixing 0.625 mL of acrylamide
solution, 1.25 mL of stacking buffer, 0.05 mL of 10% SDS
solution, 3.045 mL of water, 0.01 mL of TEMED and 0.05 mL
of 10% ammonium persulfate solution. Insert a 7 or 10-well
gel comb immediately without introducing air bubbles.
4. Add native sample loading buffer to the samples which are to
be electrophoresed. Then load them in consecutive wells while
avoiding any lateral diffusion. Electrophorese at 15–25 mA
until the dye front has reached the bottom of the gel.
Maintaining the dye front within the gel ensures retention of
all protein molecules in it (see Notes 5–7).
5. After electrophoresis, open the gel plates with a spatula and
gently transfer to a container.
6. Wash the gel with 2.5% Triton-X 100 solution two times for
30 min each followed by water several times. Under these con-
ditions, SDS from the gel is partly removed, that allows embed-
ded proteins to restore majority of their native structures which
were lost because of strong denaturing potency of SDS (see
Notes 8 and 9).
7. Incubate the gel in developing buffer for 12–24 h at 37 °C. At
this step, extraneously added proteolytic enzymes degrade the
embedded substrate and all protein components of the test
sample except the protease inhibitor(s) (see Note 10).
8. Stain the gel using 0.025% Coomassie Brilliant Blue R-250 in
40% methanol and 10% acetic acid for 3 h at 37 °C (see Notes
11–13).
9. Wash the gels with destaining solution resulting in appearance
of bands.
4 Notes
1. While making the buffer solutions, first the salt is dissolved in

approximately 1/4th of the final volume. Then, the pH is
adjusted by adding 6 M HCl in small increments. Finally the
volume is made up using water and the pH is rechecked.
2. It is recommended to wear a mask and gloves while weighing
dry acrylamide. Inhalation of acrylamide dust can be neuro-
toxic while dermal contact can cause skin irritation and red-
ness. Acrylamide in solution is less hazardous and upon
polymerization, becomes nontoxic.
3. After destaining, the body of the gel that originally contained
substrate should be colorless. This is possible only when the
substrate is degraded to very small peptides that are difficult to
be stained. Therefore, the protein used for copolymerization
of the gel should be a good substrate of the protease which is
used for digestion of the gel, e.g. collagen or gelatin (dena-
tured form of collagen) for collagenase; BSA or casein for tryp-
sin, etc. The protease must degrade the gel bound substrate
efficiently and extensively. Otherwise, the background of the
gel will appear as light to dark blue. This is the reason why
inhibitors of a protease of narrow specificity cannot be detected
in reverse zymography. In other words, inhibitors of proteases
of broad specificity like chymotrypsin, papain, bromelain,
Proteinase K, etc. and proteases like trypsin that can easily
cleave the substrates could be better detected by reverse
zymography.
4. The amount of substrate being incorporated into the gel
should be optimized. Excess substrate might lead to a residual
blue color in the background which might require excess pro-
tease for digestion and may interfere with correct interpreta-
tion of the results.
5. The duration of electrophoresis is a vital parameter which
determines adequate separation of multiple bands within the
sample. If a sample contains both high and low molecular mass
inhibitors, then visualization of both categories of bands in a
single gel needs to be carefully standardized. Low molecular
mass inhibitors might run out of the gel if the gel is run for a
longer duration. While high molecular mass inhibitors might
not sufficiently enter into the gel if the gel is run for a shorter
duration. Thus for an unknown sample, two electrophoretic
runs of different durations might be necessary for complete
evaluation of the inhibitor profile.
6. Unlike zymography, in principle the rate of migration of the
sample should not affect the electrogram. However, too fast
migration may cause trailing of protein bands. Also, a high
Fig. 2 Loading of samples. Both (a) and (b) show that overloading and under-
loading are necessary to ascertain the purity of sample
voltage can adversely affect sharpening of protein bands due to

thermal diffusion.
7. Overloading of samples might often lead to the appearance of
a broad band. Under such conditions, absence of bands of
high or low molecular weight beyond detection limit ensures
homogeneity of the preparation. However, this can be confus-
ing with respect to the number of bands actually present within
the broad band. Figure 2 shows that gradually reducing the
concentration of the sample would ultimately reveal if more
than one band is present or not. This aspect of resolution is
true for any type of gel electrophoresis.
8. In the analysis of reverse zymogram, it is assumed that the
inhibitor molecule retains its inhibitory property after electro-
phoresis in presence of SDS followed by washing with Brij-35
or Triton-X 100 to reduce the load of SDS in the gel. If the
inhibitor is structurally unstable and is unable to restore revers-
ibly its inhibitory potency, there remains a possibility that the
inhibitor is to be digested by the protease. This is a matter of
concern when the inhibitor is structurally unstable and its
native state conformation is a requirement to bind and inactive
the protease. On the other hand, if an inhibitor resists denatur-
ation in the presence of SDS, this concern does not arise.
9. In continuation to the previous consideration, it is possible
that a protein in the test sample which is not an inhibitor of
the protease but is resistant to protease digestion due to
either structural stability or absence of proteolytically sensi-
tive bonds or inaccessibility of such bonds to the protease
should appear as a band in reverse zymography. Therefore,
reverse zymography cannot distinguish between an inhibi-
tor and a protein resistant to proteolysis. In absence of
background knowledge, an inhibitor detected by reverse
zymography should be verified by independent means like
solution state enzyme assay. This fact is illustrated in Fig. 3
where lysozyme is used as the test sample. Lysozyme is
resistant to proteolytic digestion [8]. Therefore, it appears
as an inhibitor of trypsin. In reality, lysozyme is not an
Fig. 3 Lysozyme (25 μg) digested with trypsin (0.5 mg) appears as an inhibitor in

reverse zymography
inhibitor of trypsin. This limitation of reverse zymography

has been described in details in reference [9]. This indicates
that a solution state enzyme assay should be necessarily per-
formed along with reverse zymography to confirm the
inhibitory nature of a band.
10. In reverse zymography, it is assumed that the externally applied
proteases should digest the impregnated substrate along with
all proteins that are present in the test sample except the
inhibitor/s. If it happens that the relative abundance of a pro-
tein in the sample is very large and the protease is unable to
digest the protein completely under the conditions employed,
a false band may appear along with the inhibitor(s), if any, lead-
ing to wrong interpretation of the results. While optimizing
the experimental conditions, one should ensure that digestion
of susceptible proteins must be complete.
11. Ideally, the substrate copolymerized with acrylamide should
not migrate under electrophoretic conditions. However, a por-
tion of the substrate remains free and may migrate under elec-
trophoretic field according to its isoelectric point. Usually
substrates of low molecular weight migrate more, e.g., casein
(Mr. 22–23 kDa, pI 4.2–4.7) is more likely to migrate than
BSA (Mr. 67 kDa, pI 4.8). Electrophoresis of a gel copolymer-
ized with casein after staining shows two distinct regions of
different color intensities—one lighter region due to migration
of unbound substrate and the other one containing both
Fig. 4 Blank run of casein-incorporated zymogram. The gel is divided into two
sections. Region 1 is lighter than region 2 showing migration of substrate
bound and unbound substrates (Fig. 4). Thus, the extent of

migration of the substrate should be evaluated using a blank
run in absence of the test sample. After staining, if the gel
shows two distinct regions—then the concentration of the
impregnated substrate should be decreased along with modifi-
cation of the gel polymerization protocol.
12. As stated above, after staining the gel of a reverse zymography,
two regions of darker and lighter shades may appear. In that case,
the free substrate may be electrophoretically removed before
application of the test sample. Otherwise, it will be difficult to
generate a good-quality photographic presentation of the gel.
13. The Coomassie Blue solution used for staining should not
contain any precipitate or semisolid lumps. These particles
form intense spots on the gel surface that are difficult to be
destained. For uniform staining, the dye should be passed
through Whattman filter paper 1 prior usage.
Acknowledgement
K.S. is supported by fellowship from University Grants Commission,

New Delhi (UGC/NET-SRF).
References
1. López-Otín C, Matrisian LM (2007) Emerging 6. Snoek-van Beurden PAM, Von den Hoff JW
roles of proteases in tumour suppression. Nat (2005) Zymographic techniques for the analysis
Rev Cancer 7:800–808 of matrix metalloproteinases and their inhibi-
2. Rawlings ND, Tolle DP, Barrett AJ (2004) tors. Biotechniques 38:73–83
Evolutionary families of peptidase inhibitors. 7. Hawkes SP, Li H, Taniguchi GT (2010)
Biochem J 378:705–716 Zymography and reverse zymography for
3. Visse R, Nagase H (2003) Matrix metallopro- detecting MMPs and TIMPs. Methods Mol
teinases and tissue inhibitors of metalloprotein- Biol 622:257–269
ases: structure, function, and biochemistry. Circ 8. Nonaka Y, Akieda D, Aizawa T, Watanabe N,
Res 92:827–839 Kamiya M, Kumaki Y, Mizuguchi M, Kikukawa
4. Wilkesman J, Kurz L (2009) Protease analysis T, Demura M, Kawano K (2009) X-ray crystal-
by zymography: a review on techniques and pat- lography and structural stability of digestive
ents. Recent Pat Biotechnol 3:175–184 lysozyme from cow stomach. FEBS J 276:
5. Vandooren J, Geurts N, Martens E, Van den 2192–2200
Steen PE, Opdenakker G (2013) Zymography 9. Dutta S, Bhattacharyya D (2013) Reverse
methods for visualizing hydrolytic enzymes. Nat zymography alone does not confirm the pres-
Methods 10:211–220 ence of an inhibitor. Protein J 32:155–162
Chapter 12
Cell In Situ Zymography: Imaging Enzyme–Substrate

Interactions
Aastha Chhabra and Vibha Rani
Abstract
Zymography has long been used for the detection of substrate-specific enzyme activity. In situ zymography
(ISZ), an adaptation from the conventional substrate zymography, is a widely employed technique useful
for the detection, localization, and estimation of enzyme–substrate interactions in tissues. Here, we
describe a protocol to detect ‘in position’ matrix metalloproteinase (MMP) activity in cells utilizing H9c2
cardiomyoblasts as a model. This technique is primarily adopted from the method used for histological
sections and is termed as ‘Cell in situ Zymography’. It is a simple, sensitive, and quantifiable methodology
to assess the functional activity of an enzyme ‘on site/in position’ in cell culture.
Key words Cytochemistry, Dye-quenched casein, Enzymes, Fluorescent assay, Image analysis/quan-
tification, In situ zymography, Matrix metalloproteinases (MMPs), Proteolytic activity
1 Introduction
Zymography is a vividly used technique that complements other

molecular techniques for the assessment of enzyme–substrate
interactions through its ability to detect functional activity of an
enzyme. One of the well-established zymography techniques,
known as ‘Substrate Zymography’, is an electrophoresis-based
method that involves detection of substrate-specific enzyme activ-
ity as distinct bands indicating digestion of substrate in a polyacryl-
amide gel wherein the substrate is either present in the incubation
buffer (as in Superoxide Dismutase Zymography) or is incorpo-
rated in gel (as in Gelatin Zymography) [1]. However, one of the
major drawbacks of this technique concerns the loss of enzyme
localization in the cells/tissue during sample preparation and has
compelled researchers to devise an alternate method [2–4].
Thus, with a primary objective of detection and localization of
the functional activity of an enzyme, in situ Zymography (ISZ), a
variant of substrate zymography, was developed for histological
sections [5–7]. ‘In situ’ is a Latin phrase that refers to ‘in position’
133
134 Aastha Chhabra and Vibha Rani
and this technique offers the advantage to observe and estimate

the substrate-specific enzyme activity ‘on site’. The basic principle
involves digestion of substrate by the functionally active form of
enzyme present in its native location, followed by detection of this
interaction through liberation of signal from the photophore/flu-
orophore conjugated to the substrate by light or fluorescent
microscopy. Based on the nature of substrate, ISZ has been classi-
fied into two methodologies, wherein the first method utilizes a
photographic emulsion overlay containing a substrate like gelatin
while the second uses a fluorophore labeled substrate [3, 8, 9].
The modified version of the latter recommends usage of substrate
conjugated to quenched fluorophore where the enzyme activity is
a direct function of the fluorescent signal obtained on cleavage of
substrate and liberation of the fluorophore [10]. Although the
scope of this technique ranges for a broad set of enzymes, it has
essentially been used in studies pertaining to Matrix
Metalloproteinases (MMPs), a class of calcium- and zinc-dependent
endopeptidases that play a key role in extracellular matrix (ECM)
remodeling under various physiological and pathological condi-
tions [11].
Cell culture has been a valuable material of study with its appli-
cation extending to various areas of biological research. The intro-
duction of three-dimensional culture systems has advanced the ‘in
vitro’ approach further [12]. To our knowledge, very few studies
cite the use of ISZ in cell lines. Cha and colleagues have reported
assessment of gelatinolytic activity in mouse skeletal myoblasts
(C2C12) and rat cardiac myoblasts (H9c2) utilizing DQ-gelatin in
a matrix of low-melting agarose [13]. In this chapter, we explain a
simple protocol and help extend the use of in situ Zymography in
cell culture [14].
2 Materials
Make all solutions in ultrapure water by purifying de-ionized water

to attain a sensitivity of 18 MΩ cm at 25 °C. Prepare and store all
reagents at room temperature (unless indicated otherwise). Ensure
that light-sensitive steps are carried out in dark. Follow Good
Laboratory Practices (GLP) guidelines while performing the
experiment.
2.1 Cell Culture 1. Cell line: Rat heart-derived H9c2 cardiomyoblasts obtained
Components from National Centre for Cell Science (NCCS), Pune, India
(see Note 1).
2. Growth media: Dulbecco’s Modified Eagle Medium (pH 7.4)
containing 4.5 g/L glucose, 0.584 g/L l-glutamine, 0.11 g/L
sodium pyruvate, 3.7 g/L sodium bicarbonate and 2.6 g/L
HEPES, supplemented with antibiotic (100 units/mL
Cell In Situ Zymography 135
Penicillin and 100 μg/mL Streptomycin) and 10% Fetal Bovine

Serum (FBS). Prepare and filter sterilize the media with a
0.22 μm filter. Store at 4 °C (see Note 2).
3. Cover slips: Microcoverglass No. 1, 18 × 18 mm2, sterile.
4. Multi-well plate: 6-well, total well area—9.6 cm2, well vol-
ume—15.5 mL, flat-bottom, with lid, sterile.
5. Pointed forceps—sterile.
6. Humidified CO2 incubator: 5% CO2, 37 °C.
7. Methanol: HPLC grade, purity ≥99.9%.
8. 1× Phosphate-buffered saline: 0.2 g/L KCl, 0.2 g/L KH2PO4,
1.14 g/L Na2HPO4∙2H2O and 8 g/L NaCl. Weigh the com-
ponents and dissolve in 500 mL of ultrapure water. Make up
the volume to 1 L once all the salts dissolve completely.
Autoclave and keep at 4 °C for long-term storage. Pre-warm to
37 °C before use.
2.2 Cell In Situ 1. Digestion buffer: 50 mM Tris–Cl (pH 7.4), 150 mM NaCl,
Zymography 5 mM CaCl2 and 0.1% Brij-35. Weigh the components and
Components dissolve in ultrapure water. Make up the volume of buffer once
all salts dissolve completely. Add 0.1% Brij-35 (non-ionic
detergent) with a cut tip in the final solution. Use freshly pre-
pared buffer.
2. FITC-Casein: Casein fluorescein isothiocyanate type III from
bovine milk, lyophilized and salt free (see Notes 3 and 4).
3. 0.5% Agarose solution: Agarose for routine use (Molecular
Biology Reagent grade). Prepare in digestion buffer as
described in methods section (see Subheading 3.2, step 3
and Note 5).
4.
Microscope glass slide: Glass slides for microscopes,
76 × 26 × 1.35 mm3, dust-free, pre-warmed to 45–50 °C.
5. Pointed forceps—Sterile.
6. Shallow trough—Amber colored/covered with foil, 0.5–1 cm
in depth, with lid, dust-free (see Note 6).
7. Laboratory incubator: Humidified, maintained at 37 °C
8.
Standard fluorescent microscope: With FITC filter
(Excitation—485 nm; Emission—530 nm), with DAPI filter
(Excitation—364 nm; Emission—454 nm), with camera
attached to the microscope (see Note 7).
3 Methods
Carry out all procedures at room temperature unless otherwise

specified.
3.1 Cell culture 1. Follow good laboratory practices for cell culture techniques
(see Note 8). Place a sterile cover slip in a 6-well plate using
forceps and seed the suspension of H9c2 cells covering the
entire surface of cover slip. Carefully add 2 mL of growth
media per well from one corner of the well (see Note 9).
2. Gently swirl the plate to allow the cell suspension and media to
spread uniformly in the well.
3. Allow the cells to adhere to the cover slip and grow to a conflu-
ency of about 50–60% in a humidified CO2 incubator main-
tained at 37 °C and 5% CO2 (see Notes 10 and 11).
4. Give suitable treatment to the cells as per the plan of experi-
ment to be conducted.
5. On the day of assessing MMP activity, aspirate the media and
add 1 mL of 1× phosphate buffered saline (PBS, pH 7.4) per
well to wash the cells. Gently rock the plate on a gel-rocker at
30 rpm for 15 s. Remove PBS and repeat the process twice.
6. Add 600 μL methanol to each well and fix the cells by keeping
the plate at −20 °C for 15 min (see Note 12).
7. Wash the cells with 1× PBS as described in step 5 (however, at
50 rpm for 15 s) to remove traces of methanol completely.
3.2 Cell In Situ All steps concerning fluorogenic substrate must be carried in dark.
Zymography A schematic flowchart summarizes the technique briefly (Fig. 1). It
must be ensured that the conduct of the experiment is well-
controlled as suggested in Table 1.
1. Prepare fresh digestion buffer containing 50 mM Tris–Cl, pH 7.4,
150 mM NaCl, 5 mM CaCl2 and 0.1% Brij-35 (see Note 13).
Coat slide with substrate (Agarose-FITC Casein)
Place cover slip containing fixed cells on slide
Incubate slide in digestion buffer-1 hr, 37˚C, dark

Zymolytic activity
Observe slide under fluorescent microscope
Fig. 1 Graphical overview: Fluorescently labeled substrate-based cell in situ

zymography
Table 1
Appropriate controls for cell in situ zymography
Compound Class Remarks

1. Non-Metalloprotease-specific inhibitors
PMSF Serine protease No inhibition of MMP-
specific activity
Pepstatin Aspartic protease –
Leupeptin Threonine protease –
2. Metalloprotease-specific inhibitors
o-Phenanthroline Zinc chelator Inhibits Zn2+-dependent
MMP activity
EDTA Divalent cation chelator Inhibits Ca2+- and Zn2+-
dependent MMP activity
Tetracycline and its derivatives Broad spectrum antibiotic; Cycline Nonspecific MMP inhibitor
derivative
3. MMP activator
Amino-phenyl mercuric acetate Organomercurial Activates latent form of
(APMA) MMPs
4. Enzyme activity control
Trypsin (Serine protease) Positive control Digests substrate including
casein and gelatin
5. Substrate control
FITC-Albumin Negative control Nonspecific substrate for
MMP activity
2. Make 0.5 μg/mL FITC-casein solution in digestion buffer

from the stock solution of FITC-casein substrate prepared as
per Product Datasheet. Add 1 μL of FITC-casein solution
from a stock of 0.5 mg/mL to 500 μL of digestion buffer and
mix well. This step must be carried in dark and in an amber
tube (see Note 14).
3. Also, prepare 0.5% agarose solution in digestion buffer. Add
0.1 g agarose to 10 mL digestion buffer. Boil the solution to
dissolve agarose completely as indicated by a clear solution.
Allow the solution to cool down to a temperature of 45–50 °C
(see Note 15).
4. Make 1:1 solution of FITC-casein with agarose from the solu-
tions prepared in steps 2 and 3. Add 500 μL of agarose solu-
tion (prepared in step 3) to an equal volume of FITC-casein
solution (prepared in step 2) just before it begins to solidify.
Pipette up and down to thoroughly mix the solution.
5. Immediately drop 150 μL of agarose solution containing the

fluorescently labeled substrate on the center of a clean micro-
scope glass slide, pre-warmed to a temperature of 40–50 °C
6. Pick the cover slip with fixed cells (described in Subheading
3.1) from one corner using pointed forceps and quickly place
it on the microscope glass slide with the cell side facing the gel
solution.
7. Allow the agarose solution to spread evenly and solidify such
that the cover slip embeds in the gel (see Note 18).
8. Place the microscope glass slide in a shallow bottom trough
and keep the setup in an incubator set at 37 °C. Pour digestion
buffer from one corner of the trough so as to just reach the
height of the slide (see Note 19).
9. Cover the trough with a lid on top and keep the slide undis-
turbed for 1 h in dark.
10. Remove the slide from the trough and dry it with a KimWipe
(Kimberly Clark). Observe the slide under fluorescent micro-
scope in the FITC filter. Spot regions of zymolytic activity
observed as green fluorescence signal of FITC molecule
liberated from casein with its intensity as a direct function of
caseinolytic activity (see Notes 20 and 21).
11. Fix the image capture settings such as exposure, gamma,
magnification using the Control (non-treated) set and cap-
ture images of all experimental groups with the same specifi-
cations through a camera attached to the microscope
(Fig. 2a–f) (see Notes 22–24).
12. Follow the same protocol for assessing MMP activity in
presence of inhibitor, o-phenanthroline (see Note 25).
3.3 Image Analysis 1. Total fluorescence per cell can be quantified using Image J, a
and Representation widely available image processing software (http://rsbweb.
nih.gov/ij/), and be represented as a bar or box and whisker
plot (see Note 26). Open the image to be quantified in Image
J and go to ‘Analyze’ in the tool bar. Open ‘Set Measurements’
and select parameters—‘Area’, ‘Mean Gray Value’ and
‘Integrated Density’. Redirect the specifications to the selected
image and click ‘OK’.
2. Draw the boundary of the cell to be quantified in the image
using free hand selection tool and click ‘Measure’ in the
‘Analyze’ drop down list (Shortcut key—Ctrl + M).
3. Also, draw a region in the background close to the cell selected
in step 2 using free hand selection tool and click ‘Measure’ in the
‘Analyze’ drop down list to measure the background fluores-
cence. Repeat this step for 4–5 background selections per cell.
Fig. 2 Cell in situ zymography at a glance: (a) Digestion of FITC-conjugated

casein present in agarose gel observed as green fluorescent signal indicates
MMP activity in H9c2 cells. (b) Negative control shows DAPI-stained nuclei of
cells fixed on cover slip embedded in agarose gel without FITC-Casein. (c)
Negative control shows no zymolytic activity in absence of cells on the cover slip
embedded in FITC-Casein containing agarose gel. (d) Digestion of DQ Gelatin
(alternate substrate) present in agarose gel observed as a function of fluorescent
signal indicates substrate-specific gelatinolytic activity of MMPs in H9c2 cells.
(e) Fluorescent signal liberated from FITC-Casein denotes caseinolytic activity of
MMPs in H9c2 cells fixed on a cover slip embedded in a matrix of agar (alternate
for agarose). (f) Zymolytic activity in presence of MMP inhibitor, o-phenanthroline.
A major drop in fluorescent signal, in presence of a zinc chelator, signifies the
specificity of enzyme–substrate interaction
4. Now repeat steps 2 and 3 for at least 8–10 cells per image and
quantify a minimum of four images captured from different
fields for each experimental group (accounts to minimum 40
values for each group).
5. Export the data from the ‘Output’ window to a Microsoft
Excel sheet for calculations.
6. Compute the average ‘Mean Gray Value’ obtained as a mea-
sure of background signal for each selected cell (known as
‘Mean fluorescence of background’). Calculate the Corrected
Total Cell Fluorescence (CTCF), which accounts for any noise
due to background, for each cell using the formula,
CTCF = Integrated density of selected cell − (Area of selected
cell × Mean fluorescence of background).
7. A number of CTCF values will be obtained for each experimen-
tal group that can be plotted as a bar or box and whisker plot.
However, any other representation method may also be used.
4 Notes
1. This protocol is optimized utilizing H9c2 cardiomyoblasts

(rat-derived cardiac cell line) as the model for assessment of
MMP activity. However, any other adherent cell type may also
be freely used.
2. DMEM growth medium with the formulation as described in

Subheading 2 was utilized for maintenance of H9c2 cells. The
general information, characteristics, and culture method for
each cell line are detailed by the concerned cell culture reposi-
tory. Hence, the selection of the culture conditions may there-
fore be carried out in consideration with the same (e.g. Use
RPMI-1640 medium to culture Human leukemic monocytes
(U937) and T cells (Jurkat)).
3. FITC-Casein substrate is light sensitive and water soluble. It
should be stored at −20 °C to remain stable for at least 2 years.
As per manufacturer’s instructions, it is recommended to
resuspend and aliquot FITC-Casein to smaller volumes upon
arrival. Repeated freeze–thaw cycles can lead to slight increase
in the background signal and lower the sensitivity of substrate.
Details of product used in the present study—Catalog number
C0528 (Sigma Aldrich).
4. Alternate substrate—DQ Gelatin (suggested product—DQ™
Gelatin, D12054, From Pig Skin, Fluorescein Conjugate,
Thermo Fischer Scientific). It is a highly quenched, fluorescein
labeled gelatin substrate with great degree of sensitivity that
gives bright green fluorescence upon digestion by gelatinolytic
MMPs (Excitation—485 nm; Emission—530 nm) (Fig. 2d).
Interestingly, FITC-conjugated substrates for different types
of collagen are also available now.
5. Alternate gelling agent—1% agar prepared in digestion buffer
may also be used (Fig. 2e).
6. The cover of 96-well microplate or a 100 mm culture dish may
be used for this purpose.
7. It is important to view the sample using a fluorescence micro-
scope with appropriate filters. The selection of an optimal filter
requires the user to match the optical filter specifications to the
spectral characteristics of the fluorophore being employed.
Details of fluorescent microscope and image capture software
used in the current work—Olympus CKX31 (Japan) and
ProgRes Capture Pro 2.7, respectively.
8. The key for good laboratory practice in cell culture is to ensure
that all procedures are carried out to a standard that precludes
contamination by bacteria, fungi and mycoplasma as well as
cross-contamination with other cell lines. Some of the basic
precautions are suggested as follows: (a) Surface sterilize the
work space, materials and equipments with 70% ethanol before
commencement of work. (b) Ensure that the growth media
and other reagents are sterile and opened inside the hood only.
Screw the cap of reagent bottle tightly and seal the neck before
taking it out from the hood. (c) Disinfect all materials before
removing them from the hood. Also, surface sterilize the work
area with 70% ethanol and UV, after use. (d) Routinely exam-
ine the cultures and growth media for evidence of any gross
microbial contamination. (e) Clean the incubator, cabinet,
centrifuge and microscope regularly. (f) Follow appropriate
guidelines for disposal of biological waste (infectious and non-
infectious) that includes sharps contaminated with biological
waste, liquid as well as solid waste.
9. Place only one cover slip per well in the 6-well plate. It is con-
venient to use pointed forceps with a good grip for holding
and placing the cover slip. Seed the cell suspension on cover
slip in a drop by drop fashion and gently fill the well with media
from one corner rather than directly on the cover slip to pre-
vent dislodging of cells.
10. It is possible that the cells do not adhere to the cover slip—user
must confirm the cell type (suspension/adherent) and check
for cell viability since dead cells tend to round-off and detach.
11. Seed the cells at a density such that they achieve ~60% conflu-
ency at the time of experiment. A very high cell density can
lead to over-digestion and an excessively bright fluorescent sig-
nal in which cells are not distinct. This affects the image quality
and makes the quantification troublesome. Hence, it is recom-
mended to have cells at a density such that they are discrete.
12. We prefer to use chilled absolute methanol to fix the cells
before proceeding for zymography. Fixation helps to immobi-
lize the antigen while retaining the cellular and subcellular
structures. The fixative and protocol for cell fixation may be
modified as per the properties of the cell line being used.
13. Brij-35 is a non-ionic light detergent that ensures enzyme sta-
bilization and minimizes the risk of nonspecific interactions.
14. FITC-casein is diluted in only half the volume of digestion
buffer (1 μL FITC-casein from stock of 5 mg/mL in 500 μL
digestion buffer) such that its effective concentration in the
substrate–agarose (1:1) mixture remains 0.5 μg/mL.
15. Agarose is dissolved in only half the volume of digestion buffer
(0.1 g agarose in 10 mL buffer) such that its effective concen-
tration in FITC-casein containing gel (1:1) solution remains
0.5%. Do not add the fluorophore-conjugated substrate to the
agarose solution until it cools down to a temperature of ~45 °C
(i.e. temperature below which it begins to solidify).
16. Cut the end of the tip or use a wide-orifice tip for a smooth
flow while dropping the gel solution on glass slide. This will
also help avoid air bubbles on the slide.
17. Pre-warming of microscope glass slide allows just enough time
to drop the gelling agent on the slide and place the cover slip
over it before the solution solidifies. A little delay prevents the
cover slip from embedding in the gel and thus, increases the
chance of its displacement when incubated in the digestion
buffer.
18. The agarose solution spreads evenly by capillary action and
covers the entire surface area of the cover slip as it is placed
over the gel mixture. Care must be taken while placing the
cover slip to ensure that there is no air bubble between the
cover slip and gel solution present on the glass slide.
19. Care must be taken to avoid displacement of the cover slip
from its position during this step. It is thus suggested, to first
place the trough containing slides in the incubator and then
carefully add the digestion buffer in the trough. Adding a suf-
ficient volume of digestion buffer in the trough ensures that
the setup does not dry during the incubation period.
20. Take out the slide carefully from the trough such that the cover
slip does not displace from its position during handling. Dab
the excess liquid over a piece of KimWipe/tissue paper before
setting up the slide for imaging.
21. The fluorescein label on FITC-Casein substrate is highly
quenched. Upon digestion by MMPs present in the cells, the
substrate is cleaved into smaller peptides, thus, terminating the
quenching of the fluorescent label.
22. Capture the same field in case of dual staining (i.e. simultane-
ous execution of cell in situ zymography (Green—Fluorescein-
conjugated substrate) with nuclear staining (Blue—DAPI) so
that well-defined overlays may be created. Additionally, the
cells must also be viewed in the bright field.
23. Nuclei of cells may be stained with a commonly used dye,
DAPI (4′,6-diamidino-2-phenylindole), which binds to
nucleic acids. It is a light-sensitive, water-soluble stain with
an excitation/emission maxima at 358/461 nm. Stock solu-
tion of DAPI must preferably be aliquoted and stored at
−20 °C. However, the solution may be stored at 2–8 °C for
a short term. Preparation of DAPI solution: Resuspend
DAPI in storage solution (Stock concentration 1 mg/mL;
Working concentration: 50 ng/mL). The storage solution
may also be utilized for all serial dilutions. Storage solution
(Tris Buffer Solution): 10 mM Tris–HCl pH 7.4, 10 mM
EDTA pH 8, 100 mM NaCl. Caution: DAPI is a known
mutagen and hence, precaution must be taken while han-
dling it. Safe disposal of dye in compliance with safety regu-
lations is recommended.
24. In case of very weak/strong fluorescent signal, the user must
troubleshoot based on parameters including substrate concen-
tration, MMP activation, incubation period and temperature.
25. o-Phenanthroline (1,10-phenanthroline monohydrate) is a

metalloproteinase inhibitor that chelates divalent metals like
zinc and iron. It is soluble in methanol and can be prepared at
a stock concentration of 200 mM. The stock remains stable at
−20 °C for months. Effective concentration: 1–10 mM. The
inhibitor is added to the digestion buffer used for making the
gel solution as well as that utilized for incubation (Fig. 2f).
26. Image J is one of the most commonly used image processing
software. However, other softwares like Image-Pro Plus
(Media Cybernetics) may also be used. Additionally, we sug-
gest one useful link to make a box and whisker plot (http://
boxplot.tyerslab.com).
Acknowledgement
This work was supported by the research grant awarded to Dr.

Vibha Rani by the Department of Science and Technology
(DST), Government of India (SR/FT/LS-006/2009) and
Department of Biotechnology (DBT), Government of India
(BT/PR3978/17/766/2011).
References
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ize endogenous protease activity in tissue Integr Physiol 156:518–522
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7. George SJ, Johnson JL (2010) In situ zymog- Rani V (2012) Cell in situ zymography: an in
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Microscopic localization of active proteases by Anim 48:463–468
Part IV
2D Zymography
Chapter 13
Examination of Gelatinase Isoforms in Rodent Models

of Acute Neurodegenerative Diseases Using
Two-Dimensional Zymography
Shanyan Chen, Fanjun Meng, Zhenzhou Chen, Zhe Qu, Jiankun Cui,
and Zezong Gu
Abstract
Pathological activation of gelatinases (matrix metalloproteinase-2 and -9; MMP-2/-9) has been shown to
cause a number of detrimental outcomes in neurodegenerative diseases. In gel gelatin zymography is a
highly sensitive methodology commonly used in revealing levels of gelatinase activity and in separating the
proform and active form of gelatinases, based on their different molecular weights. However, this meth-
odology is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity
can be regulated at transcriptional and/or post-translational levels under in vivo conditions resulting in
alternation of their isoelectric focusing (IEF) points. In this chapter, we describe an advanced methodol-
ogy, termed two-dimensional zymography, combining IEF with zymographic electrophoresis under non-
reducing conditions to achieve significant improvement in separation of the gelatinase isoforms in both
cell-based and in vivo models for acute brain injuries and neuroinflammation.
Key words Matrix metalloproteinase-2 and -9, Gelatinases, Two-dimensional zymography, Post-
translational modification isoforms, Neurodegeneration
1 Introduction
Matrix metalloproteinases (MMPs) are members of a family of 26

zinc-dependent endopeptidases that contain structurally similar
hemopexin, propeptide, and catalytic domains. They are so-called
metalloproteinases due to their dependence on metal ions, particu-
larly Zn2+ and Ca2+, as cofactors in the enzymatic active site [1, 2].
MMPs regulate homeostasis of the extracellular matrix (ECM) by
proteolysis of its components, such as collagen, laminin, fibronec-
tin and collagens, as well as many non-matrix bioactive molecules
Fanjun Meng (Deceased)
147
148 Shanyan Chen et al.
[3]. MMPs are needed for modulating interactions between cells

during development and for repair, including tissue remodeling,
cell migration, wound healing, angiogenesis, and embryogenesis
[4, 5]. However, dysregulation of MMPs are also known to cause
progression of damage in various diseases. In neurological diseases,
excessive activation of MMPs is implicated in multiple sclerosis,
HIV-associated dementia, spinal cord injury, traumatic brain injury
(TBI), amyotrophic lateral sclerosis (ALS), and stroke [6–10].
In the MMP family, MMP-2 and MMP-9 are also called gela-
tinases due to their ability to degrade gelatin, the denatured form
of collagen. Reportedly, pathological activation of gelatinases has
been shown to cause a number of detrimental outcomes, including
disruption of the blood–brain barrier (BBB), hemorrhage, neuro-
nal apoptosis [11], and brain damage in ischemic stroke [8] and
traumatic brain injury (TBI) [10]. MMP-9 (Gelatinase B) activity,
in particular, is significantly elevated in humans after stroke
[12–14]. High plasma MMP-9 concentration in the acute phase of
cerebral infarct is considered as an independent surrogate of hem-
orrhagic transformation in all stroke subtypes [15]. Our group and
others have shown that aberrant MMP-9 proteolytic activity is
associated with an increase in BBB permeability and neuronal
injury after acute cerebral ischemia [8, 16–18].
MMPs are expressed in the cells and secreted into the sur-
rounding ECM as inactive zymogens. The Zn2+ ion in the active
site of the enzyme is blocked by a cysteine residue located in the
pro-domain of the MMP peptide. MMP enzyme becomes proteo-
lytically active only when the Zn2+ ion in active site is exposed by
dissociation of the binding between the cysteinyl sulfhydryl in the
propeptide domain and the catalytic Zn2+ ion. This mechanism of
activation is referred to as the “cysteine switch” [19]. The removal
of the cysteine residue from the MMP active site can be achieved
by cleavage of the pro-domain, resulting in a lower molecular
weight active MMP [20]. The activity of MMPs can also be regu-
lated by the levels of post-translational modifications, including
S-nitrosylation, S-glutathiolation, glycosylation, sialylation, and
phosphorylation [8, 21, 22]. It has been reported that MMPs
could be activated by the pro-oxidant species peroxynitrite
(ONOO−) without removal of the inhibitory propeptide domain.
ONOO− may react with the cysteine thiol within the PRCGVPD
sequence of the propeptide domain by S-glutathiolation, resulting
in an increase in proteolytic activity. It suggests that full-length
form of the MMP may also be proteolytically active [23–25].
Phosphorylation is another post-translational modification playing
a significant role in regulating MMPs. It has been demonstrated
that phosphorylation of MMP-2 (Gelatinase A) by inflammatory
factor protein kinase C increases its activity [26].
In-gel zymography (IGZ) was first introduced in 1978 to study
plasminogen activators [27]. In an IGZ experiment, hydrolytic
2D Zymography Differentiates Gelatinase Isoforms 149
enzymes are separated by their molecular weights and detected by their

ability to degrade the substrate copolymerized in the separating gel of
SDS-PAGE. Briefly, after separation by non-reducing SDS-PAGE gel,
the sodium dodecyl sulfate (SDS) is replaced by a non-ionic detergent
with a lower critical micelle concentration. This step allows the enzymes
to be partially refolded to their active forms; these enzymes are previ-
ously unfolded by SDS ion. Next, the gel is incubated in a buffer with
essential cofactors in certain temperature, which allows the reactivated
enzymes to degrade the copolymerized substrate. Proteolytic activity is
detected by light zones on a dark background following staining pro-
cedures [28]. With IGZ, information that can be obtained includes:
proteolytic activity of specific enzyme, molecular weights, and the pres-
ence of covalent complexes or fragments in complex biological sam-
ples. However, accumulated evidences show that complex
post-translational modification is involved in the activation of MMPs
without changing molecular weights by the removal of the inhibitory
pro-peptide domain [24, 26, 29]. Therefore, identification of different
isoforms of gelatinases based on molecular weights after electrophore-
sis is not sufficient in complex biological samples.
We complemented conventional IGZ with an additional
dimension of protein separation by in-gel IEF to achieve signifi-
cant improvement in separating the enzymatic isoforms due to
their various pI values [30]. In this so-called 2D zymography
method, gelatinase isoforms with charge differences can be sepa-
rated in the first IEF dimension, followed by the conventional
zymography. We have found in LPS-stimulated microglial cells and
ischemic/TBI mouse brain tissue, this extra IEF dimension is par-
ticularly useful for identifying different MMP-9 isoforms [30]. We
also found the MMP-9 pI values were shifted in 2D zymography
after treatment with alkaline phosphatase, suggesting the signifi-
cance of phosphorylation in MMP-9 activation [30]. Therefore,
2D zymography is demonstrated as an effective method to separate
isoforms of gelatinases with different post-translational modifica-
tions in acute brain injuries.
2 Materials
2.1 Brain Extraction 1. Brain Extraction Buffer: 50 mM Tris, pH 7.6, 150 mM NaCl,
and Gelatinase 5 mM CaCl2, 0.05% Brij35, 1% Triton X-100.
Purification 2. Gelatin Sepharose 4B.
3. Elution Buffer: 8 M urea, 4% CHAPS.
2.2 Isoelectric 1. Rehydration Buffer: 8 M urea, 4% CHAPS, 0.5% IPG buffer
Focusing (see Note 1).
2. SDS Equilibration Buffer: 50 mM Tris–HCl, pH 8.8, 6 M
urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue.
2.3 Gelatin 1. Separating gels: 0.375 M Tris–HCl, pH 8.8, 10% acrylamide/

SDS-PAGE Gel bis-acrylamide (29:1), 0.1% SDS, 0.05% ammonium persulfate
(APS), 0.005% TEMED, 10% glycerol, 0.1% gelatin.
2. Stacking gels: 0.125 M Tris–HCl pH 6.8, 4% acrylamide/bis-
acrylamide (29:1), 0.1% SDS, 0.05% APS, 0.001% TEMED.
3. Agarose Sealing Mixture: 0.5% agarose, 0.002% bromophenol
blue in 1× electrophoresis running buffer.
4. Electrophoresis Running Buffer: 2.9 g Tris-base, 14.4 g gly-
cine, 1 g SDS, doubly distilled H2O to 1 L.
5. Renature Buffer: 2.5% Triton X-100.
6. Developing Buffer: 50 mM Tris–HCl pH 7.6, 0.2 M NaCl,
5 mM CaCl2, 0.2% Brij35.
7. Coomassie Blue Staining Solution: 0.2% Coomassie brilliant
blue R-250, 40% ethanol, 10% acetic acid.
8. Destaining solution: 40% ethanol, 10% acetic acid.
3 Methods
In this section, we describe how to examine isoforms of MMP-2

and MMP-9, both in lysates prepared from brain samples, and
from conditioned media of BV-2 microglial cultures.
3.1 Protein The filament-induced transient middle cerebral artery occlusion

Extraction (MCAo) or TBI is performed as described previously [8, 10, 30–32].
and Gelatinase Mice are sacrificed with an overdose of isoflurane and transcardially
Purification perfused with saline to remove intravascular blood. Mouse brains
are rapidly collected.
Immortalized mouse BV-2 microglial cells are originally from
Dr. R. Donato (University of Perugia, Italy) [33]. BV-2 cells are
maintained in DMEM (Dulbecco’s Modified Eagle Medium) con-
taining 5% heat-inactivated FBS (fetal bovine serum) at 37 °C in a
saturated humidity atmosphere containing 95% air and 5% CO2. At
70–80% confluence, BV-2 cells are starved with no serum medium
for 4 h and treated with 100 or 500 ng/mL LPS in the condi-
tioned medium for 16 h as described previously [30].
1. Weigh the brain tissues.
2. Add extraction buffer with proteinase inhibitors. Volume of
extraction buffer (μL) = weight of sample (mg) × 6.
3. Homogenize the brain tissues on ice with a pestle in a micro-
centrifuge tube. Keep the homogenate on ice for 20 min.
4. Centrifuge (17,000 × g) the homogenate for 20 min at
4 °C. Collect the supernatant.
5. To measure protein concentration, 10 μL of each sample is

added into 190 μL of ddH2O, making a 20-fold dilution.
Perform BCA assay following manufacturer’s manual.
6. Make all samples from step 4 to the same concentration
according to the results of BCA assay.
7. Wash the Gelatin Sepharose 4B with extraction buffer for three
times.
8. After the last wash, carefully aspirate the supernatant. Add
protein samples from step 6 to the Gelatin Sepharose 4B. For
cell-
based studies, add conditioned media to the Gelatin
Sepharose 4B.
9. Rotate the samples in tubes slowly at 4 °C overnight to purify
gelatinases.
3.2 Gelatinase 1. Wash Gelatin Sepharose 4B with extraction buffer three times
Elution and then discard supernatant.
2. Add 75 μL of elution buffer to each tube and rotate the tubes
for 40 min at room temperature.
3. Centrifuge (17,000 × g) the samples for 30 min at room tem-
perature. Collect the supernatant (75 μL) for the following
steps.
3.3 First 1. Mix the samples and rehydration buffer. Volume of this mix-
Dimensional ture depends on the length of IPG strips. For 7-cm strips,
Separation: Isoelectric 125 μL mixtures are applied.
Focusing 2. Distribute the mixture in strip holders evenly. Lay the IPG
strips on the mixture.
3. Cover the IPG strips with mineral oil to minimize evaporation.
4. IPG strips are swelled with the protein samples for 12 h under
50 V (active rehydration).
5. Proteins are separated by IEF (PROTEAN IEF Cell, Bio-Rad)
using the following conditions with: 250 V (rapid) for 250
voltage-hours (V⋅h); 500 V (rapid) for 500 V⋅h; 1000 V (rapid)
for 1000 V⋅h; 5000 V (linear) for 10,000 V⋅h and 5000 V
(rapid) for 20,000 V⋅h.
6. Equilibrate the IPG strips with the SDS equilibration buffer
twice, each time 15 min with shaking.
7. Wash the IPG strips with running buffer before loading to the
second-dimensional gels.
3.4 Second- 1. First, prepare separating gels (30 mL for four gels): mix 3 mL
Dimensional of ddH2O; 3 mL of 1% gelatin; 6 mL of 50% (v/v) glycerol;
Separation: Gelatin 10 mL of 30% acrylamide–bis-acrylamide; 7.5 mL of separating
SDS-PAGE gel buffer stock (1.5 M Tris buffer, pH 8.8); 300 μL of 10%
(w/v) SDS; 150 μL of 10% (w/v) APS. Degas the solution for
approximately 5 min. To speed up the process, dissolve the gel-

atin with warm water.
2. Add 15 μL of TEMED to the separating gel solution to initiate
polymerization. Immediately, pipette approximately 6.2 mL of
the separating gel solution into the gel casting chamber (Mini-
PROTEAN Tetra Cell, Bio-Rad). Avoid formation of bubbles.
3. Carefully overlay the separating gel solution in the cassette
with ddH2O using a syringe. Do not disturb the surface of the
separating gel solution.
4. Let the assembly stand for at least 1 h at room temperature to
polymerize. Polymerization is complete when discrete lines of
separation can be noted between the gels and the water
overlay.
5. Prepare stacking gels: decant the overlay water from the sepa-
rating gels. Immediately, add 6 μL of TEMED to the stacking
gel solution, swirl rapidly and pipet the solution onto the top
of the polymerized separating gels until it reaches the top of
the front plate.
6. Rapidly, insert the appropriate combs into the liquid stacking
gels, making sure that no bubbles remain trapped under the
combs. Let the stacking gel polymerize at room temperature
for 30 min.
3.5 Running 1. Apply the equilibrated IPG strips to the second-dimensional

and Developing gels carefully.
of Gelatin SDS-PAGE 2. Add 1 mL of agarose sealing mixture over the IPG strips to
Gels cover them completely.
3. Run the gels at constant voltage (120 V) until the 37 kDa
band of the prestained maker runs out of the gels.
4. Carefully remove the gels from the cassette and place it in plas-
tic tray containing 100 mL of renaturing buffer. Incubate the
gels for 30 min at room temperature with gentle agitation for
three times. Replace the renaturing buffer with 100 mL of
developing buffer. Wash the gels for 30 min at room
temperature.
5. Replace the developing buffer with 100 mL of fresh develop-
ing buffer. Incubate the gels at 37 °C for about 18 h in the
incubator (see Note 2).
6. Stain the gels with Coomassie blue staining solution for 4 h at
room temperature, agitating on a rotary shaker.
7. Destain the gels with destaining solution for 15–30 min at
room temperature, agitating on a rotary shaker. Replace the
solution with water when the clear lanes and spots are revealed
(see Fig. 1) [30].
Fig. 1 2D zymography reveals gelatinase isoforms from conditioned medium of

LPS-stimulated microglial BV-2 cells. MMP-9 isoforms were visualized as a seri-
ous transparent spots with pI values ranging at 3.5–7 and molecular weight at
100 kDa; MMP-2 was visualized as a single spot with pI value between 5 and 6,
and molecular weight at 65 kDa
4 Notes
1. No reducing reagents (e.g. DTT) are allowed for 2-D

zymography.
2. Developing time can be adjusted based on the amount and the
activity of the gelatinases in different samples.
Acknowledgments
This work was supported in part by the American Heart Association

National Scientist Development award (09SDG2260983), The
Dana Foundation, The National Football Leagues (NFL) Charities
Foundation, The University of Missouri Mizzou Advantage One
Health One Medicine Program and the Department of Pathology
Research funds to Z.G.
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dimensional zymography differentiates gelatin- Hadass O, Lehmidi T, Blair GJ, Lee M, Chang
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Chapter 14
Two-Dimensional Zymography of Proteases from Steatotic

Duck Liver
Jeff Wilkesman, María Fernanda Padrón, Liliana Kurz,
and Hervé Rémignon
Abstract
Protease activity present in liver cells with steatosis can be electrophoretically characterized.
Zymographic techniques allow semi-quantitative results, successfully detecting cathepsin and metallo-
protease activity using polyacrylamide gels copolymerized with gelatin and quantified by densitometry.
By using specific inhibitors, the identity of the proteases can be confirmed. 2D zymography allows the
determination of both Mr. and pI of the metalloprotease and cathepsin activity present in the homog-
enates. The analysis of liver proteases activities in force fed ducks may elucidate the mechanisms behind
steatosis development.
Key words Electrophoresis, MMP2, Cathepsins, Steatosis, Zymography
Abbreviations
2DE Two dimensional electrophoresis
2DZ Two dimensional zymography
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
IEF Isoelectric focusing
MMP Matrix metalloproteases
PAG Polyacrylamide gel
PAGE Polyacrylamide gel electrophoresis
pI Isoelectric point
PMSF Phenylmethyl sulfonyl fluoride
SDS Sodium dodecyl sulphate
TCA Trichloroacetic acid
157
158 Jeff Wilkesman et al.
1 Introduction
The accumulation of triglycerides in the hepatic cells is known as

fatty liver or steatosis, usually as the result of a rich carbohy-
drates diet force feeding [1]. In palmipeds, the liver is the place
where most of the fat is synthesized and can be stored in case of
excessive production. In force fed birds, due to the large amount
of carbohydrates brought by the daily diet, a huge quantity of fat
is produced and stored, leading to the development of a nutri-
tional steatosis of the liver [2]. In any case, a steatotic liver in
ducks is not a pathological organ, but one that presents an
enhanced metabolic activity [1].
In the regulation of a wide range of biological processes are
present many proteolytic activities, as many possess high specificity
associated with the hydrolysis of the peptide bond [3, 4]. Numerous
techniques are used to determine the presence of proteases in bio-
logical samples. The application of electrophoresis represents a
simple alternative in the study of many types of proteases and its
inhibitors [5, 6]. Zymography is an important tool for the detec-
tion of proteolytic activity on an electrophoretic gel matrix,
employing a protein substrate [7].
In this chapter, we describe the detection of proteolytic pro-
files present in hepatic cells under steatosis, employing 1D and 2D
zymography. To accomplish this, an appropriate extraction proce-
dure was performed. For quantitative purposes, it is advised to
measure in parallel the total proteolytic activity by some other
method, e.g. photometry.
2 Materials
All chemicals used were of analytical grade, and deionized water

was used. As main equipment an electrophoretic chamber is needed
(many commercial providers offer different types and models)
along with a power supply.
2.1 Extraction 1. Extraction buffer without inhibitors: 50 mM Tris–HCl pH 7.5,

Buffers 150 mM NaCl, 1 mM CaCl2.
2. Extraction buffer selective for metalloproteases: 50 mM Tris–
HCl pH 7.5, 150 mM NaCl, 1 mM CaCl2, 10 μg/mL leu-
peptin, 1 mM PMSF.
3. Extraction buffer selective for cathepsins: 50 mM Tris-HCl
pH 7.5, 150 mM NaCl, 1 mM CaCl2, 20 mM EDTA.
2.2 Electrophoresis 1. 1.5 M Tris pH 8.8.

Stock Solutions 2. 0.5 M Tris pH 6.8.
2D Zymography of Duck Liver Proteases 159
3. 40% Acrylamide.
4. 2% Bis-acrylamide.
5. 5% gelatin stock solution: weigh 0.5 g and dissolve in hot water
until 10 mL. Aliquot 1 mL fractions and keep refrigerated.
6. 10% APS: 0.0520 g in 520 μL. Prepare fresh.
7. TEMED.
8. 4× sample buffer: 200 mM Tris–HCl pH 6.8, 4% (w/v) SDS,
40% (v/v) glycerol, 0.02% (w/v) bromophenol blue.
9. Running buffer: 25 mM Tris–HCl, 192 mM glycine, 0.1%
(w/v) SDS.
2.3 Staining 1. Coomassie staining solution: 0.125% (w/v) Coomassie Brilliant

Solutions Blue R-250, 40% (v/v) 2-propanol, 10% (v/v) acetic acid.
2. Coomassie staining solution for zymograms: 0.5% (w/v)
Coomassie Brilliant Blue R-250, 40% (v/v) 2-propanol, 10%
(v/v) acetic acid.
3. Destaining solution: 40% (v/v) 2-propanol, 10% (v/v) acetic
acid.
2.4 2D 1. Rehydration buffer: 8 M urea, 1% (w/v) CHAPS, 10 mM

Electrophoresis DTT, 0.25% (v/v) Biolytes 3-10.
2. Equilibration buffer I: 8 M urea, 0.375 M Tris–HCl pH 8.8,
2% (w/v) SDS, 20% (v/v) glycerol, 130 mM DTT.
3. Equilibration buffer II: 8 M urea, 0.375 M Tris–HCl pH 8.8,
2% (w/v) SDS, 20% (v/v) glycerol, 270 mM IAA.
4. Agarose solution: 1% agarose, 0.025% bromophenol blue.
2.5 Zymography 1. Metalloprotease renaturing buffer: 100 mM glycine pH 8.0,

Solutions 2.5% (v/v) Triton X-100.
2. Cathepsin renaturing buffer: Tris–HCl 50 mM pH 7.4, 20%
(v/v) glycerol.
3. Metalloprotease activation buffer: 100 mM glycine pH 8.0.
4. Cathepsin activation buffer: 0.1 M sodium acetate pH 5.5,
1 mM EDTA, 2 mM DDT.
2.6 Other Standards 1. Bovine serum albumin solution: BSA (1 g/L), weigh 1 mg pro-
tein and dissolve in 1 mL extraction buffer without inhibitor.
2. Molecular weight standards (commercially available, for SDS-
PAGE and for zymograms).
3. Bradford protein determination kit.
3 Methods
3.1 Biological 1. Collect the liver samples from ducks or other source [we stud-
Samples ied Muscovy ducks (Cairina moschata)].
2. Treat the sample according to the protocol reported by Awde [8].
3. Briefly, livers are removed from carcasses, weighted, and sam-
ples are harvested, immediately frozen in liquid nitrogen and
stored at −80 °C until analysis (see Note 1).
3.2 Protein 1. Weigh ~400 mg tissue and homogenize at 4 °C in a potter

Extraction with a specific buffer.
2. Choose a specific extraction buffer, either: (a) without inhibi-
tors; (b) selective for metalloproteases, or (c) selective for
cathepsin.
3. Centrifuge suspension at 10,000 × g for 20 min at 4 °C. If
necessary remove the fat cake due to lipids precipitation and
redo the centrifugation.
4. Resulting supernatant is collected and either used immediately
or alternately stored at −20 °C until assayed.
3.3 Protein 1. Determine the total amount of protein according to the

Quantification Bradford method [9] (see Note 2).
2. Perform a calibration curve by triplicate employing bovine
serum albumin as standard protein.
3. Assay samples by duplicate, measuring absorbance at λ595 nm.
3.4 SDS–PAGE 1. This electrophoresis is a modified procedure described by

Laemmli [10]. Mind that non-reducing conditions are present,
i.e., absence of 2-mercaptoethanol and samples are not heated.
2. Prepare solutions in order to polymerize 10% resolving gels
and 5% stacking gel (Table 1).
3. Polymerize gels.
4. Dissolve ~30 μg protein in 4× sample buffer and apply samples
into the well.
5. Apply the appropriate amount of molecular weight standards
(consult the info-sheet of your product).
6. Add running buffer to the chamber.
7. Begin electrophoresis by setting on the power supply at 90 V
for the first 15 min, and then increase to 120 V. Perform the
run at 4 °C (see Note 3).
8. Run until the blue front reaches the bottom of the gel.
9. Remove gels from the glasses, and place them in container
with Coomassie staining solution.
Table 1
Resolving and stacking gel preparation
5%
10% SDS-PAGE 10% Zymography Stacking
resolving gel resolving gel gel
Stock component Volume (mL)

H2O 3.35 3.15 1.2
1.5 M Tris pH 8.8 2.5 2.5 –
0.5 M Tris pH 6.8 – – 0.5
40% Acrylamide 2.5 2.5 0.2
2% Bis-Acrylamide 1.6 1.6 0.1
5% Gelatin – 0.2 –
10% APS 0.05 0.05 0.01
TEMED 0.005 0.005 0.002
Final volume 10 10 2
(for two mini-gels)
10. Remove staining solution and place the gel in destaining solu-
tion until patterns are observed.
11. Digitalize the gels with a transilluminator and save images as
jpeg files.
12. Determine MW by using a calibration curve with the standard
proteins, and relate the log MW vs. relative mobility.
3.5 2DE 1. Add rehydration buffer to an aliquot of liver extract containing

between 100 and 400 μg of total protein such that the final
volume is under 150 μL (see Note 4). Centrifuge briefly.
2. Place the IPG over the sample and leave 30 min for
absorption.
3. Add 500 μL mineral oil.
4. Program the focusing run according to Table 2.
5. Submit strips to rehydration under active conditions: 50 V,
20 °C, 15 h, without pause after rehydration, and with 50 μA/
strip.
6. Take notice that the final V × h displayed on the screen should
be >10,000 V⋅h.
7. During the last focusing step, prepare the equilibration buffer
I and II according to Table 3 (see Note 5).
8. Also during this time, polymerize a 10% resolving gel (Table 1)
and a 10% zymogram without stacking gel.
Table 2
Program conditions for focusing
Step Total time (h) Voltage (V)

Rehydration 15 50
Focusing (rapid ramp) 3.5 50 → 4000
Slope down (linear ramp) 5 4000 → 500
Table 3
Composition of equilibration buffers
Equilibration Equilibration
buffer I buffer II
Final concentration of
the component Amount Amount Stock
6 M urea 1.8 g 1.8 g 60 g/mol
0.375 M Tris pH 8.8 1.25 mL 1.25 mL 1.5 M
2% SDS 1 mL 1 mL 10%
20% Glycerol 1 mL 1 mL 100%
2% DTT 0.1 g –
2.5% IAA – 0.125 g
BrPhBlue 25 μL 1%
Final volume 5 mL 5 mL –
9. After focusing, equilibrate strips for 20 min in equilibration

buffer I. Place strips in the special equilibration/rehydration
tray and add 2.5 mL buffer per strip.
10. Now, equilibrate strips in equilibration buffer II.
11. Finally, wash strips briefly with water and then place one of
them over the 10% polyacrylamide gels, and the other over the
zymogram containing 0.1% gelatin substrate.
12. Seal the strip by overlaying it with warm agarose solution.
13. Run gels as described under Subheading 3.4, step 6.
14. When run is finished, stain the gel without substrate with
Coomassie staining solution. Depending on the type of prote-
ase to be detected, proceed as stated under Subheading 3.8 for
the zymogram.
3.6 1DZ 1. Prepare a 10% SDS-PAGE (Table 1) copolymerized with gela-

for Metalloproteases tin (see Note 6).
2. Pre-run the gel in running buffer at 4 °C for 20 min at 125 V.
3. Apply ~50 μg total protein per lane.
4. Run the system at 4 °C for 120 min at 125 V.
5. After run, incubate gels twice in renaturing buffer for 30 min
each.
6. Discard solution and place gel in activating buffer at 37 °C for
18 h.
7. Finally, stain gels with Coomassie staining solution for
zymograms.
8. Add destaining solution until pale bands under a deep blue
background appear.
3.7 1DZ 1. According to Platt et al. [11] for the case of cathepsins, polym-
for Cathepsins erize 10% PAG (Table 1) containing 0.1% gelatin (see Note 7).
2. Prepare the samples in sample buffer. Set up the chamber and
add running buffer.
3. Run at 100 V, 4 °C, until the blue front reaches the bottom of
the gel (~2 h).
4. Once the run is completed, wash gel twice for 15 min with
0.1 M sodium acetate.
5. Place gel in cathepsin renaturing buffer under mild agitation
overnight.
6. Now incubate gels with cathepsin activation buffer at 37 °C for
18 h.
7. Afterward, soak gels briefly in water (dd) and stain as described
in Subheading 3.6, step 7. Figure 1 shows the protein profile of
a duck liver sample in a 10% SDS-PAGE and a 10% zymogram.
3.8 2DZ 1. After the 2DE run, place gels in 1% Triton-X 100 solution, for
1 h.
2. Incubate in a suitable activation buffer, depending on the pro-
tease activity to be identified.
3. Wash and incubate gels with the same solutions used for the
1DZ. An example of a 2DE and 2DZ is given in Fig. 2, for the
cathepsin analysis.
4. Perform densitometric analysis for p/ and MW determina-
tion. Spot intensities can be correlated with proteolytic activi-
ties (see Note 8) [12–15].
a b
250
150
100
75
50
37
25
20
15
10
Fig. 1 10% SDS PAGE (a) and 10% zymogram (b) of duck liver homogenate extracted for cathepsin detection
75
50
37
25
20
3 pl 10
B
75
50
37
25
20
Fig. 2 2DE and 2DZ examples. First dimension run with IEF strips from pH 3–10.
Second dimension run in SDS-PAGE 10%. (a) 2DE of the protein extract from
duck liver. (b) 2DZ of the cathepsin activity
4 Notes
1. Before performing any experiments, consult the bioethical

regulations of your laboratory, institution or country. The
experiment described here fully complies with legislation on
research involving animal subjects according to the European
Communities Council Directive of November, 24 1986
(86/609/EC), the Venezuelan Law of Science & Technology
(2010) and the Venezuelan Bioethic Code (2010). In the case
of France, French Investigators must be certificated by the
French governmental authority for carrying out these experi-

ments [16–18].
2. Total protein content (μg/μL) in the sample needs to be deter-
mined before submitting samples to electrophoresis, in order
to know the amount of sample (in our case ~30 μg) needed for
correct visualization of the activity on the zymograms.
3. The chamber may be placed directly inside a refrigerator, or a
cold room.
4. For two strips, for example, we prepare a final volume of
250 μL. Our total protein content in the extract was 7.6 μg/
μL. We took a 20 μL aliquot and combined it with the rehydra-
tion buffer. Each strip was covered with 125 μL solution.
5. Equilibration buffer I and II are freshly prepared.
6. We have also used 7.5% SDS-PAGE to obtain a better band
resolution. The adequate % will depend on the MW of the
protease and its polypeptide profile.
7. We have also used 12% SDS-PAGE to obtain a better band
resolution. The adequate % will depend on the MW of the
protease and its polypeptide profile.
8. Proteolytic activity can be measured by the Kunitz method
[19].
Acknowledgments
We thank INP-ENSAT for supporting the stay of Dr. Wilkesman at

GenPhySE, Université de Toulouse, as well as the Consejo de
Desarrollo Científico y Humanísitico de la Universidad de Carabobo,
Venezuela, for partial funding of this research (CDCH-AM-030-11,
CDCH-187-2015). The authors thank the staff of GenPhySE lab
for technical assistance and Dr. Molette for her useful comments.
References
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Gong DQ (2011) Gene expression profile in by zymography: a review on techniques and
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Sci 90:107–117 6. Shiju J, Sudhakaran P (2003) Changes in the
2. Hermier D, Salichon MR, Guy G, Peresson R activity of matrix metalloproteinases in regen-
(1999) Differential channelling of liver lipids in erating rat liver after CCl4-induced injury.
relation to susceptibility to hepatic steatosis in Indian J Biochem Biophys 40:324–329
the goose. Poult Sci 78:1398–1406 7. Snoek P, Von den Hoff J (2005) Zymographic
3. Bugg T (2012) Introduction to enzyme and techniques for the analysis of matrix metallo-
coenzyme chemistry, 3rd edn. John Wiley & proteinases and their inhibitors. Biotechniques
Sons, UK, pp 77–102 38:73–83
4. Rawlings N, Barrett A (1993) Evolutionary 8. Awde S, Marty-Gasset N, Wilkesman J, Rémignon
families of peptidases. Biochem J 290:205–218 H (2013) Proteolytic activity alterations resulting
from force-feeding in Muscovy and Pekin. Poult 14. Natale M, Caiazzo A, Ficarra E (2016) A novel
Sci 92:2997–3002 Gaussian extrapolation approach for 2-D gel
9. Bradford MB (1976) A rapid and sensitive electrophoresis saturated protein spots.
method for the quantitation of micrograms quan- Methods Mol Biol 1384:203–211
tities of protein utilizing the principle of protein- 15. Chen S, Meng F, Chen Z, Tomlinson BN,
dye binding. Anal Biochem 72:248–254 Wesley JM, Sun GY et al (2015) Two-
10. Laemmli UK (1970) Cleavage of structural dimensional zymography differentiates gelatin-
proteins during the assembly of the head of ase isoforms in stimulated microglial cells and
bacteriophage T4. Nature 227:680–685 in brain tissues of acute brain injuries. PLoS
11. Platt M, Randall A, Hanjoong J (2006) One 10(4):e0123852. doi:10.1371/journal.
Laminar shear stress inhibits cathepsin L activ- pone.0123852
ity in endothelial cells. Arterioscler Thromb 16. Código de Bioética y Bioseguridad (2008)
Vasc Biol 26:1784–1790 3era Edic, pp 1–63
12. Brauner JM, Groemer TW, Stroebel A, Grosse- 17. European Communities Council Directive
Holz S, Oberstein T, Wiltfang J, Kornhuber J, (1986) (86/609/EC). On line: http://ec.
Maler JM (2014) Spot quantification in two europa.eu/food/fs/aw/aw_legislation/
dimensional gel electrophoresis image analysis: scientific/86-609-eec_en.pdf. 13 July 2016
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sentation of a novel compound fitting algo- Innovación. (2010) Gaceta Oficial N° 39.575.
rithm. BMC Bioinformatics 15:181 16 Dec 2010
13. Morris JS, Gutstein HB (2016) Detection and 19. Kunitz M (1947) Crystalline soybean trypsin
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Part V
Special Zymography Cases

Chapter 15
Simultaneous Detection of Activity and Relative Molecular

Mass of Adenylate Kinases After SDS-PAGE and Blotting
Silvia Ravera and Isabella Panfoli
Abstract
Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases, which play a pivotal role in the
energetic metabolism. At the present, nine isoforms are known. AKs catalyze the following reversible reac-
tion: ATP + AMP ↔ 2 ADP, even though isoform 3 uses GTP instead ATP. For many years, the activity of
AKs has been detected only after native polyacrylamide gel separations, i.e. in the absence of sodium
dodecyl sulfate or methanol. In this work, we report the possibility to detect the activity of the isoforms
able to use ATP as substrate, directly onto gel or nitrocellulose sheets, after denaturing SDS-PAGE and
electroblotting. This method is innovative because it allows to determine simultaneously the activity and
the molecular weight of AKs, especially onto nitrocellulose where bands are sharper, thanks to absence of
protein diffusion.
Key words Adenylate kinase, SDS-PAGE, Electroblotting
1 Introduction
Adenylate kinase (AK; EC 2.7.4.3) isoenzymes are involved in

energy metabolism and homeostasis of cellular adenine nucleotide
ratios in different intracellular compartments [1–3]. AKs belong to
the cellular nucleotide synthetic machinery, catalyzing the revers-
ible reaction AMP + MgATP ↔ ADP + MgADP [2]. Nucleotides
make up the structure of nucleic acids and are also important in cell
metabolism either as a source of chemical energy or as activated
intermediates in many biosynthetic pathways, in cell signaling and
as components of coenzymes [2]. The preferred substrate of all
AKs is AMP and their main phosphate donor is ATP, although
some can phosphorylate other substrates and use other phosphate
donors, such as GTP [2].
At the present, in vertebrates, nine AK isoforms are known:
ubiquitous and cytoplasmic AK1, AK7 and AK8 (all around
21 kDa) [2, 4]; AK2, located in the outer compartment of mito-
chondria (26 kDa) [5]; AK3, mitochondrial matrix GTP:AMP
169
170 Silvia Ravera and Isabella Panfoli
phosphotransferase (26 kDa) [6]; AK4, expressed in the pyramidal

hippocampus cells (21 kDa) [7]; brain-specific AK5, expressed
during differentiation (21 kDa) [8] and AK6 (21 kDa) expressed
at nuclear level [9]. AK has also been identified in bacteria, and is
called AK9 (22 kDa) [10]. Until now, the AKs have been catego-
rized into two major subgroups: the short-type AK1 group and the
long-type AK2–AK3 group, based on their Mr. [11]. Metabolically,
the several isoforms can be sorted into two groups: those that uti-
lize ATP (AK1, 2, 4, 5, 6, 7, 8, and 9), and those that use GTP as
a phosphate group donor (AK3) [2].
AKs activity can be assayed using several techniques. Principally,
it is monitored spectrophotometrically at 340 nm, on the basis of
the enzyme coupling method, which follows either the NADP
reduction or the NADH oxidation [12]. However, this method
does not allow distinguishing the several isoforms, because most of
them catalyze the same reaction, using ATP.
Another way to assay the AKs activity is represented by zymog-
raphy, performed without denaturing agents [13]. However, also
in this case is not possible to discriminate the different isoforms.
More recently, isoelectric focusing has been proposed to determine
the intracellular distribution of the AK isoforms in mammalian tis-
sues, discriminating only two AK isoforms, AK1 and AK2, on the
basis of the different isoelectric points [14].
Here, we report a method to detect AKs activity after denatur-
ing SDS-PAGE and/or blotting on nitrocellulose (NC), allowing to
detect concurrently both the activity and the Mr. It is important to
note that, even though, in this method, the sample is denatured by
heat (95–100 °C for 5 min) and β-mercaptoethanol is present, the
AKs activity can be restored, because the enzyme is stable to heat
[15] and does not contain Cys residues in the active site (as reported
on Uniprot Data Bank: http://www.uniprot.org/uniprot/
P00568). However, to detect AKs activity after SDS-PAGE is funda-
mental to perform the run at very low amperage (20 mA) and to
remove the Sodium dodecyl sulfate (SDS) from the gel, with casein
buffer for on gel assay [1]. However, it is not applicable to observe
the activity of the isoform 3, the GTP:AMP phosphotransferase.
Moreover, the different isoforms can be identified either by using a
specific antibody after the activity detection on NC, or by perform-
ing the assay on a specific cellular fraction, which contains only one
of the cited isoforms.
2 Materials
Prepare all the solutions using ultrapure water (prepared by purify-

ing deionized water to attain a sensitivity of 18 MΩ cm at 25 °C)
and analytical grade reagents. Take the appropriate safety precautions
for chemical hazards in carrying out the experiments. Diligently fol-
low all waste disposal regulations when disposing waste materials.
Adenylate Kinase Zymography 171
2.1 Sample 1. In this work, we used liver and muscle, withdrawn from male,
Preparation 10 weeks old ICRCD1 mice anaesthetized with ether and
decapitated (see Note 1). However, this method is apt for sev-
eral types of samples, from bacterial cultures to mammalian
tissues. Homogenize the samples in 0.25 M sucrose, 5 mM
HEPES buffer, 1 mM EDTA, pH 7.2.
2. Prepare 50 mL homogenization buffer, by mixing 4.27 g
sucrose, 0.06 g HEPES and 0.03 g EDTA with 40 mL ultra-
pure water. When the salts are completely dissolved, add 5 M
HCl until pH 7.2 is reached (see Note 2), and make up the
solution to 50 mL, with ultrapure water. The solution can be
conserved at −20 °C for several months.
2.2 Electrophoresis For this analysis, a polyacrylamide gel at 14% was employed.
Polymerize gel using Mini PROTEAN® Tetra Cell Casting Module
and Mini PROTEAN® 3 System glass plates. Perform the electro-
phoresis using Mini-PROTEAN® tetra cell system and a universal
power supply. For gel polymerization, prepare the following
solutions:
1. Resolving gel buffer composed by 1.5 M Tris–HCl pH 8.8,
10 mM EDTA, 14 mM SDS. For 0.5 L buffer, dissolve 91.2 g
Tris and 1.5 g EDTA in 400 mL ultrapure water. When the salts
are dissolved, adjust the pH to 8.8 with 5 M HCl (see Note 2).
Make up the solution to 0.5 L and add 1.5 g SDS (see Note 3).
Store an aliquot at 4 °C, and stock the remainder at −20 °C.
2. Stacking gel buffer, composed by 0.5 M Tris–HCl, pH 6.8
(corresponding to 30.4 g for 0.5 L ultrapure water), 10 mM
EDTA and 14 mM SDS. Prepare the buffer as described for
the resolving gel buffer. Store an aliquot at 4 °C, and stock the
remaining buffer at −20 °C.
3. 30% Acrylamide/Bis-acrylamide solution (see Note 4). The
ratio among acrylamide and bis-acrylamide is 37.5:1 (2.7%
cross-linker) (see Note 5).
4. Ammonium persulfate: 10% solution in water. Store the solu-
tion at −20 °C in the dark.
5. N, N, N′, N′-tetramethyl-ethylenediamine (TEMED) (see
Note 6).
6. SDS-PAGE running buffer 5× (stock solution): 0.25 M Tris–
HCl, pH 8.3, 1.5 M glycine, 11 mM EDTA and 0.1%
SDS. Prepare the solution by dissolving 30 g Tris, 144 g gly-
cine and 3.38 g EDTA in 1 L ultrapure water. Once salts are
dissolved, add 5 g of SDS (see Note 3). The solution can be
stored at 4 °C for several months.
7. Sample buffer 4× (stock solution): 25% stacking gel buffer
(pH 6.8), 10% SDS, 25% 2-mercaptoethanol, 0.1% bromophenol
blue, 0.012 M EDTA and 1.5 M sucrose. For 20 mL, mix 5 mL
stacking gel buffer with 8 g sucrose, 0.25 mL 2-mercaptoethanol,
0.02 g bromophenol blue, and 0.07 g EDTA in a 50 mL conical
flask. Made up to 20 mL with ultrapure water. Add 1.6 g SDS and
mix gently, to avoid bubble formation. 0.5 mL aliquots were pre-
pared and stored at −20 °C (see Note 7).
2.3 Blotting 1. Nitrocellulose membranes with 0.45 μm pore size.

2. Wypall*X-60 reinforced paper.
3. Blotting buffer: 0.025 M Tris, 0.192 M glycine, and 20% meth-
anol. Prepare 1 L of solution, adding 14.4 g glycine to 200 mL
methanol plus 600 mL ultrapure water, in a 1 L beaker. Adjust
the pH to 8.3 with Tris (see Note 8) and make up to 1 L with
ultrapure water. Store the solution at 4 °C (see Note 9).
4. Perform the blotting using Mini Trans-Blot Module (see Note
10) with a magnetic stirrer and a universal power supply.
2.4 In Gel Detection 1. 1 M Tris–HCl pH 8.6: dissolve 12.1 g Tris in 80 mL ultrapure
of AKs Activity water; when the salts are completely dissolved add 5 M HCl,
until pH 8.6 is reached (see Note 2) and make up the volume
to 100 mL with ultrapure water. The solution can be stored at
4 °C for several months.
2. 1 M KCl: dissolve 7.45 g KCl in 100 mL ultrapure water. The
solution can be stored at 4 °C for several months.
3. 1 M MgCl2: dissolve 9.52 g MgCl2 in 100 mL ultrapure water.
4. 2% agarose: dissolve 1 g agarose in 50 mL ultrapure water (see
Note 11) and heat it in a microwave oven, until complete
melting.
5. SDS removal buffer, containing 2% casein (w/v), 0.04 M Tris–
HCl pH 8.6 and 2 mM EDTA, pH 8.0. For each gel, prepare
100 mL of solution, dissolving 2 g casein and 0.058 g EDTA
in 80 mL ultrapure water, add 4 mL Tris–HCl pH 8.6 and
make up to 100 mL with water (see Note 12).
6. 20% Ethanol solution: dissolve 20 mL ethanol in 80 mL ultra-
pure water.
2.4.1 Reaction Mixture The reaction mixture contains 0.5 M Tris–HCl pH 8.6, 4 mM
to Assay AKs Activity, AMP, 4 mM ATP, 10 mM phosphoenolpyruvate (PEP), 1.2 mM
Following ADP Formation NADH, 0.167 M KCl, 0.2 M MgCl2.
For one gel, 15 mL solution is needed. Dissolve 0.02 g AMP,
0.033 g ATP, 0.025 g PEP and 0.0119 g NADH in 7.5 mL 1 M
Tris–HCl pH 6.8 stock solution, 0.25 mL 1 M KCl stock solu-
tion, 3 mL MgCl2 stock solution and 4.25 mL ultrapure water
(see Note 13).
For this assay, a solution of purified pyruvate kinase (PK) + lactate
dehydrogenase (LDH) is needed.
2.4.2 Reaction Mixture The reaction mixture contains 0.2 M Tris–HCl pH 8.0, 0.2 M
to Assay AKs Activity, MgCl2, 5 mM ADP, 100 mM glucose, 1.6 mM NADP, 0.2 mM
Following ATP Formation phenazine methosulfate (PMS), 0.5 mM nitro blue tetrazolium
(NBT). For one gel, 15 mL of solution are needed. Dissolve
0.032 g ADP, 0.27 g glucose, 0.018 g NADP, 9 mg PMS and
6 mg NBT in 3 mL 1 M Tris–HCl pH 6.8 stock solution, 3 mL
MgCl2 stock solution and 11 mL ultrapure water (see Note 14).
For this assay, a solution of purified hexokinase (HK) + glu-
cose 6 phosphate dehydrogenase (G6PD) is needed.
2.5 Detection of AKs 1. 1 M Tris–HCl pH 7.4 stock solution: dissolve 12.1 g Tris in
Activity 80 mL ultrapure water; when the salt is completely dissolved,
on Nitrocellulose add 5 M HCl until pH 7.4 is reached (see Note 2) and make
up the volume to 100 mL with ultrapure water. This solution
can be stored at 4 °C for several months.
2. Tris buffered saline (TBS):1.5 M NaCl, 0.1 M Tris–HCl
pH 7.4. For 0.5 L, dissolve 87.66 g NaCl in 100 mL Tris–HCl
pH 7.4 and make up to 0.5 L with ultrapure water.
3. Blocking solution: 5% milk in TBS (see Note 15).
4. The reaction mixtures are the same described for the in-gel
detection (see Subheading 2.4).
3 Methods
3.1 Sample All operations were carried out at 4 °C.

Preparations
1. Skeletal muscle S9 fraction: homogenate 3 g of mouse muscle
by Potter-Evehjem system in 10 mL homogenization buffer.
Centrifuge the sample for 10 min at 500 × g, discard the pellet
and centrifuge the supernatant for 20 min at 20,000 × g.
Retain the resulting supernatant and store it at −80 °C.
2. Liver nuclei enriched fractions: homogenate 5 g of mice liver
by Potter-Evehjem system in 27 mL of homogenization buffer
(see Note 16). Centrifuge the sample at 800 × g for 10 min.
Resuspend the resulting pellet in 10 mL homogenization buf-
fer and repeat the centrifugation. Resuspend the pellet in ultra-
pure water. Aliquot sample and store it at −80 °C.
3. Liver mitochondria enriched fractions: centrifuge the superna-
tant resulting by the first centrifugation of nuclei isolation at
15,000 × g for 20 min. Dissolve the pellet in 5 mL homogeni-
zation buffer and repeat the centrifugation. Resuspend the pel-
let in ultrapure water. Aliquot sample and store it at −80 °C.
4. Brain homogenates: Homogenate 5 g of mice brain by Potter-
Evehjem system in 27 mL homogenization buffer (see Note 16).
Aliquot sample and store it at −80 °C.
5. For each sample, measure the protein concentration. For

example, using the Bradford method [16], the protein concen-
tration should be assessed around 20–30 mg/mL. It is impor-
tant to load the same amount of total protein in gel.
3.2 SDS-PAGE Considering the low Mr. of AKs, we have used a 14% acrylamide
Electrophoresis gel. For 1 gel it is necessary to prepare 10 mL resolving gel and
2.5 mL stacking gel.
1. Prepare the resolving gel mixing: 4.67 mL 30% acrylamide,
2.5 mL resolving buffer, 50 μL APS, 6 μL TEMED and
2.77 mL ultrapure water in a 15 mL conical flask (see Note
17). Cast gel within a 7 × 8.3 × 0.1 cm3 gel cassette. Allow
space for stacking the gel and gently overlay with isobutanol
2. Wait 20–30 min to allow the polymerization of resolving gel
(see Note 20).
3. Discard isobutanol.
4. Prepare the stacking gel by mixing 0.256 mL 30% acrylamide,
0.5 mL stacking buffer, 40 μL APS and 2 μL TEMED and
1.7 mL ultrapure water in a 10 mL conical flask (see Note 16).
Insert a 10-well gel comb immediately without introducing air
bubbles.
5. Wait 10–20 min to allow the polymerization of stacking gel
(see Note 19).
6. For each sample, use 40 μg of total proteins. Warm the sample
at 95–100 °C for 5 min with 1/4 of total volume of Sample
Buffer 4× solution.
7. Perform gel run at 20 mA/gel for 120–150 min with Running
Buffer at 4 °C or over ice (see Note 21).
Alternatively, a precasted gel commercially available can be
used, provided it has the same percentage of acrylamide that we
recommend.
3.3 Detection Before proceeding with the AKs activity assay, SDS must be
of Adenylate Kinase removed from the gel.
Activity on Gel
1. Prepare the SDS removal buffer and warm it at 37 °C.
2. Following electrophoresis, separate the glass plates with a spat-
ula. Gel remains on one of the glass plates. Rinse gel with water
and transfer it carefully to a plastic container bearing the same
dimensions as the gel.
3. Wash the gel with the pre-heated SDS removal buffer for 1 h
at 37 °C, with gentle agitation on a shaker rotating at
50–60 rpm, changing the buffer each 15 min (see Note 22).
4. Prepare one of the two assay solutions, put it on the gel, add
15 mL of the 2% agarose solution and incubate at 37 °C in the
dark.
5. Check the activity band formation: (a) If the ATP formation is
followed, a violet band will appear on a light yellow back-
ground (see Fig. 1, Panel B). (b) If the ADP formation is fol-
lowed, the gel must be observed using a UV lamp. The AK
activity appears as a dark band on a fluorescent background (see
Fig. 1, Panel A) (see Note 23).
6. When the bands are visible, reaction can be stopped by adding
25 mL 20% ethanol solution.
3.4 Detection 1. After the electrophoresis run, separate the glass plates, using a
of Adenylate Kinase spatula. Gel remains on one of the glass plates. Rinse gel with
Activity After SDS- water and transfer it carefully to a container filled with blotting
PAGE buffer.
and Nitrocellulose- 2. Cut a nitrocellulose membrane of the gel size of and rinse it
Blotting twice in blotting buffer.
3.4.1 Blotting 3. Transfer the proteins on nitrocellulose membrane by the classic
electroblotting technique: 400 mA, for 1 h, at 4 °C, in blotting
buffer (see Note 24).
3.4.2 AKs Activity After transfer, wash twice the nitrocellulose membrane with 10 mL
Detection TBS.
1. Incubate the nitrocellulose with the blocking solution for 1 h,
at room temperature with a gentle agitation on a shaker rotat-
ing at 100–150 rpm.
2. Wash twice the nitrocellulose with 10 mL TBS.
3. Incubate the nitrocellulose with one of the two assay solutions,
in the dark at 37 °C.
4. Check the activity band formation. (a) If the ATP formation is
followed, a violet band will appear on a light yellow background
Fig. 1 On-gel assay of adenylate kinase activity, following ATP (Panel A) or ADP (Panel B) formation. The loaded
samples are: Lane 1, skeletal muscle cytosolic fraction, containing the isoform 1 (21 kDa); Lane 2, liver mitochondria-
enriched fraction, containing the isoform 2 (26 kDa); Lane 3, liver nuclei-enriched fraction, containing isoform 6
(21 kDa); Lane 4, brain homogenate, containing isoform 5 (21 kDa). The different isoforms have been identified
considering the subcellular fraction employed. The figure is representative of at least ten experiments
Fig. 2 Assay of AKs for ATP formation onto NC. The loaded samples are: Lane 1,
skeletal muscle cytosolic fraction, containing the isoform 1 (21 kDa); Lane 2, liver
mitochondria-enriched fraction, containing the isoform 2 (26 kDa); Lane 3, liver
nuclei-enriched fraction, containing isoform 6 (21 kDa); Lane 4, brain homoge-
nate, containing isoform 5 (21 kDa). The different isoforms have been identified
considering the subcellular fraction employed. The figure is representative of at
least ten experiments
(see Fig. 2). (b) If the ADP formation is followed, the nitrocel-

lulose membrane must be observed in a close cabinet, equipped
with an UV lamp. The AK activity appears as a dark band on a
fluorescent background.
5. In both cases, stop the reaction, discarding the assay solutions
and rinse the membrane with TBS.
4 Notes
1. Approval to conduct the experiments was obtained from the

Italian ministry of Health in compliance with animal care
requirements that are requested by Italian law (law D.L.
27.1.1992 n. 116, in agreement with the Council Directive
2010/63EU of the European Parliament and the Council of
22 September 2010 on the protection of animals used for sci-
entific purposes). All efforts were made to minimize the num-
ber of animals used and their suffering.
2. It is important that 5 M HCl is added drop by drop, to avoid
a sudden change in pH below the required one. Moreover, it
is recommended that the solution is prepared with a magnetic
stirrer.
3. It is preferable to add SDS at the end to avoid the formation of
excess foam during mixing.
4. Considering that acrylamide is a carcinogenic and neurotoxic
molecule, the use of a ready-made solution minimizes the dan-
ger of its use.
5. This type of solution was chosen because it is ideal for high
molecular weight protein electrophoresis.
6. Considering the pungent smell, the solution must be used
under fume hood.
7. SDS precipitates at 4 °C. Therefore, the lysis buffer needs to be

warmed prior to use.
8. Normally, 4–5 g Tris are necessary to reach a pH 8.3. The Tris
quantity varies on the basis of quality and purity of glycine and
on the starting pH of ultrapure water.
9. The solution can be reused several times, as long as it remains
transparent. When the solution color varies to yellow, it must be
discarded. It is important that the solution remains at 4 °C until
its use, to help to maintain a low temperature during the blot.
10. This apparatus contains also a plastic tank to be filled with ice
to keep the temperature low during the blot. For convenience,
we recommend to fill the tank with water and freeze it. This
will render temperature control more effective.
11. This stock solution can be preserved at 4 °C for 1 week.
However, in these conditions agarose becomes solid. Therefore,
it is necessary to warm-up the solution in a microwave oven,
until complete melting of agarose, letting it cool up to 45 °C
just prior use.
12. The solution must be freshly prepared each time and heated at
37 °C to dissolve completely the casein.
13. The solution must be prepared freshly each time, just prior to
the assay.
14. The solution must be prepared freshly each time, just prior to
the assay, and maintained in the dark.
15. The solution must be prepared freshly each time.
16. Sample is considered well homogenized when the tissue debris
are no longer visible. To improve the quality of the homoge-
nate, it is recommended to filter sample through gauze.
17. TEMED and APS must be added last, just before transferring
the solution in the gel cast system, to avoid that the solution
polymerizes in the conical flask.
18. This overlay prevents contact with atmospheric oxygen (which
inhibits acrylamide polymerization), moreover it helps to level
the resolving gel solution.
19. During the resolving gel polymerization, it is necessary to
avoid the formation of air bubbles.
20. To accelerate the gel polymerization, the system can be put in
an incubator at 37 °C.
21. To maintain AKs activity during SDS page, it is fundamental to
perform the run with less than 20 mA. Moreover, is also
important to keep the system at low temperature, around 4 °C.
22. This step is fundamental to assay the AKs activity after SDS-
PAGE. AKs activity could not be recovered on-gel when
detection was performed without previous casein washing.

The casein buffer removes the SDS, improving the extent of
enzyme renaturation.
23. In this case, the band visualization is more difficult with respect
to that following ATP formation. For this reason, the signal
may appear most blurred.
24. Blotting must be performed in cold room (at 4 °C) or in an
ice-bath, to maintain temperature low.
References
1. Ravera S, Calzia D, Panfoli I, Pepe IM, Zhang C, Su XD (2005) The crystal structure
Morelli A (2007) Simultaneous detection of of human adenylate kinase 6: an adenylate
molecular weight and activity of adenylate kinase localized to the cell nucleus. Proc Natl
kinases after electrophoretic separation. Acad Sci U S A 102:303–308
Electrophoresis 28:291–300 10.
Amiri M, Conserva F, Panayiotou C,
2. Panayiotou C, Solaroli N, Karlsson A (2014) Karlsson A, Solaroli N (2013) The human
The many isoforms of human adenylate kinases. adenylate kinase 9 is a nucleoside mono- and
Int J Biochem Cell Biol 49:75–83 diphosphate kinase. Int J Biochem Cell Biol
3. Notari L, Morelli A, Pepe IM (2003) Studies on 45:925–931
adenylate kinase isoform bound to disk mem- 11. Fukami-Kobayashi K, Nosaka M, Nakazawa
branes of the rod outer segment of bovine ret- A, Go M (1996) Ancient divergence of long
ina. Photochem Photobiol Sci 2:1299–1302 and short isoforms of adenylate kinase:
4. Kubo S, Noda LH (1974) Adenylate kinase of molecular evolution of the nucleoside mono-
porcine heart. Eur J Biochem 48:325–331 phosphate kinase family. FEBS Lett 385:
5. Tomasselli AG, Noda LH (1980) Mitochondrial 214–220
ATP:AMP phosphotransferase from beef heart: 12. Bergmeyer HU (1983) Method of enzymatic
purification and properties. Eur J Biochem analysis. Verlag Chemie, Germany
103:481–491 13. Bockelmann W, Ritter H (1968) Tissue vari-
6. Tomasselli AG, Schirmer RH, Noda LH ability of the phosphotransferases adenylate
(1979) Mitochondrial GTP-AMP phos- kinase (EC: 2.7.4.3.) and pyruvate kinase (EC:
photransferase. 1. Purification and properties. 2.7.1.40.) Hum Genet 6:373–376
Eur J Biochem 93:257–262 14. Kurokawa Y, Takenaka H, Sumida M, Oka K,
7. Yoneda T, Sato M, Maeda M, Takagi H (1998) Hamada M, Kuby SA (1990) Multiforms of
Identification of a novel adenylate kinase system mammalian adenylate kinase and its monoclo-
in the brain: cloning of the fourth adenylate nal antibody against AK1. Enzyme 43:57–71
kinase. Brain Res Mol Brain Res 62:187–195 15. Calzia D, Panfoli I, Ravera S, Dazzi E,
8. Van Rompay AR, Johansson M, Karlsson A Gandolfo S, Pepe IM, Vergani L, Morelli A
(1999) Identification of a novel human adenyl- (2009) Structural modification of proteins by
ate kinase. cDNA cloning, expression analysis, direct electric current from low voltage.
chromosome localization and characterization J Biochem Mol Toxicol 23:309–317
of the recombinant protein. Eur J Biochem 16. Bradford MM (1976) A rapid and sensitive
261:509–517 method for the quantitation of microgram quan-
9. Ren H, Wang L, Bennett M, Liang Y, Zheng tities of protein utilizing the principle of protein-
X, Lu F, Li L, Nan J, Luo M, Eriksson S, dye binding. Anal Biochem 72:248–254
Chapter 16
Silver-Stained Fibrin Zymography: Separation of Proteases

and Activity Detection Using a Single Substrate-Containing
Gel
Chang-Su Park, Dae-Ook Kang, and Nack-Shick Choi
Abstract
Silver-stained fibrin zymography for separation of protease bands and activity detection using a single sub-
strate gel was designed. The method takes advantage of the nano-scale sensitivity of both zymography and
silver staining. After sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a gel
containing fibrin (protease substrate), the gel was incubated in enzyme reaction buffer and the zymogram
gel was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein
substrate had been degraded. The molecular sizes of proteases were accurately determined.
Key words Protease, Electrophoresis, Silver staining, Nano-scale detection, Coomassie Brilliant Blue,
Zymography, Binding mode
1 Introduction
Proteases (EC 3.4._._) are essential constituents of all living cells,

including prokaryotes, fungi, plants, and animals, and play a criti-
cal role in cell growth and differentiation [1]. Proteases have many
commercial applications, particularly in the food, leather, deter-
gent, pharmaceutical, diagnostics, waste management, and silver-
recovery industries [2].
Substrate-containing polyacrylamide gel electrophoresis
(PAGE) (zymography) is valuable for the analysis of proteolytic
systems. Zymography is a versatile two-stage technique involving
protein separation by sodium dodecyl sulfate (SDS) PAGE, fol-
lowed by detection of enzyme activity in polyacrylamide gels
under non-reducing (no treatment of reducing agent DTT or
β-mercaptoethanol) and non-denaturing (no heating) condi-
tions. Protease activity in zymograms is visualized as a clear band
of proteolysis that appears against the dark-blue background
[after Coomassie Brilliant Blue (CBB) staining] of undigested
179
180 Chang-Su Park et al.
protein substrate [3–5]. The technique is simple and sensitive,

and protease activities may be quantified; the approach is thus
widely used to study enzyme function in complex mixtures and to
search for specific enzyme types. In the study, we compare silver
staining of zymogram gels with protease detection using CBB
[6]. Silver staining of polyacrylamide gels allows most proteins to
be detected in the nanogram range and the technique is thus
100-fold more sensitive than CBB staining. Using this method,
we succeed in overcoming some limitations of zymography.
These are, first, differences in protease migration patterns when
various enzymes are examined; second, poor resolution of high
molecular weight proteins; and third, problems with detection of
proteases that remain near the origins of substrate-containing
gels (binding mode) [7, 8].
2 Materials
Prepare all solutions using distilled water and analytical grade

reagents. We used the Bio-Rad Mini-Protean III apparatus mini-
gel system (Bio-Rad, Hercules, CA, USA) due to the savings in
materials and time. SDS-PAGE was carried out based on the
Laemmli system [9].
2.1 Sample 1. Two crude culture supernatants from Bacillus sp. strains were
Preparation used (see Note 1). If necessary the samples were concentrated
using organic solvent [10, 11] (see Note 2).
2.2 SDS Fibrin 1. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Weigh 181.7 g
Polyacrylamide Gel Tris and add to 800 mL distilled water. Mix and adjust pH
Components with concentrated HCl (see Note 3). Add distilled water to a
total volume of 1 L. Store at 4 °C.
Tris and add to 400 mL distilled water. Mix and adjust pH
with concentrated HCl. Add distilled water to a total volume
of 500 mL. Store at 4 °C.
3. Bovine fibrinogen and bovine thrombin: 1.2% fibrinogen solu-
tion and 10 NIH units thrombin in each 1 mL distilled water
(see Note 4).
4. 30% acrylamide (acrylamide:bis-acrylamide 29.2:0.8): Weigh
29.2 g of acrylamide and 0.8 g of bis-acrylamide and add dis-
tilled water about 80 mL. Stir until completely dissolved and
make up to 100 mL with distilled water and filter through a
0.45 μm filter (see Note 5). Store in a dark bottle at 4 °C.
5. 10% SDS: Weigh 10 g and add distilled water to make
100 mL. Store at room temperature.
Silver-Stained Fibrin Zymography 181
6. 10% ammonium persulfate (APS): Dissolve 0.1 g APS in 1 mL

distilled water (see Note 6).
7. Electrophoresis buffer (Tris-glycine buffer): 25 mM Tris base,
192 mM glycine, 0.1% SDS, pH 8.3 (see Note 7). Store at
room temperature.
8. Zymogram SDS sample buffer (5×): 0.5 M Tris–HCl, 10%
SDS, 20% glycerol, and 0.1% bromophenol blue (BPB),
pH 6.8. Store at −20 °C (see Note 8).
9. 1% BPB: Dissolve 0.1 g BPB in 10 mL distilled water. Filter to
remove impurity. Store at room temperature.
2.3 Zymogram Gel 1. Renaturation buffer: 50 mM Tris–HCl (pH 7.4) with 2.5%
Activation (v/v) Triton X-100 (see Note 9). Store at room temperature.
2. Zymogram reaction buffer: 30 mM Tris–HCl, 200 mM NaCl,
0.02% NaN3, pH 7.4 (see Note 10). Store at room
temperature.
2.4 Coomassie 1. Coomassie staining solution: 0.1% (w/v) Coomassie Blue

Brilliant Blue Staining R-250, 40% methanol, 10% acetic acid, 50% distilled water.
Components Store at room temperature and can be stored for several weeks.
2. Coomassie destaining solution: 40% methanol, 10% acetic acid,
50% distilled water. Store at room temperature and can be
stored for several weeks.
2.5 Silver Staining 1. Fixing solution: 40% ethanol, 10% acetic acid, 50% distilled
Components water. Store at room temperature and can be stored for several
weeks.
2. Sensitizing solution (0.02% sodium thiosulfate): Dissolve
0.02 g sodium thiosulfate in 100 mL distilled water.
3. Staining solution (0.2% silver nitrate): Dissolve 0.2 g silver
nitrate (Bio-Rad) in 100 mL distilled water and add 74 μL 35%
formaldehyde just before use (see Note 11).
4. Developing solution (6% sodium carbonate): Dissolve 6 g
sodium carbonate in 100 mL distilled water and add both
50 μL 35% formaldehyde and 4 mL sensitizing solution (item
2) just before use (see Note 11).
5. Stop and storage solution: The same as fixing solution (item 1).
Store at room temperature for several weeks.
3 Methods
Carry out sample preparation and electrophoresis at 4 °C (cold

room) and silver staining and other procedures at room
temperature.
3.1 Sodium Dodecyl 1. Prepare an Erlenmeyer flask and mix the fibrin containing sep-
Sulfate Fibrin aration gel stated in Table 1 (see Note 5). Degas it for 10 min
Zymogram Gel to get rid of dissolved oxygen. Add SDS, APS, and TEMED
Electrophoresis (12%) (Table 1), and cast gel within a mini-gel cassette
(7.25 × 10 × 0.10 cm3). Allow space for stacking gel and over-
lay the cast gel with isobutanol or distilled water (see Note 12).
2. After polymerization of the separating gel pour off the isobu-
tanol or distilled water and wash the space with distilled water
(see Note 13).
3. Prepare an Erlenmeyer flask and mix the stacking gel stated in
Table 1 (see Note 5). Degas it for 10 min to get rid of dissolved
oxygen. Add SDS, APS, and TEMED (Table 1). Pipet stacking
gel solution onto separating gel and insert comb immediately
without trapping air bubbles.
Table 1
Preparation of substrate-containing SDS zymogram gel
Volume
Component (mL)
Separating gel (12%)
Acrylamide-bisacrylamide mixture 4.0
1.5 M Tris–HCl 2.5
Bovine fibrinogen (0.012 g/mL) 1.0
Bovine thrombin (1 NIH unit/mL) 0.1
Distilled water 2.2
Degas
10% SDS 0.1
10% APS 0.1
TEMED 0.04
Total volume 10.0
Stacking gel (5%)
Acrylamide-bisacrylamide mixture 0.33
0.5 M Tris–HCl 0.25
Distilled water 1.38
Degas
10% SDS 0.02
10% APS 0.02
TEMED 0.002
Total volume 2.0
4. After polymerization of the stacking gel remove the comb carefully

and rinse the wells with electrophoresis buffer (Tris-glycine buffer)
using a microsyringe to remove unpolymerized acrylamide.
5. Two crude culture supernatants from soil microorganisms
(see Note 1) were diluted fivefold with zymogram sample buf-
fer. Samples were centrifuged and loaded to the two types of
gel (SDS gel and zymogram gel) under non-reducing (no
treatment of reducing agent DTT or β-mercaptoethanol) and
non-denaturing (no heating) conditions (see Note 14).
6. The gel was electrophoresed at 12 mA constantly in the cold
room or in ice till the dye front has reached the bottom of the
separating gel (see Note 15).
3.2 Renaturation 1. After electrophoresis, disassemble the glass plate sandwich and
and Enzyme Activation remove the stacking. Rinse the gel with distilled water and
of Fibrin Zymogram transfer carefully to a container.
Gel 2. Soak the gel with renaturation buffer for 30 min with gentle
agitation (see Note 9).
3. Rinse 10 min with distilled water three times to remove Triton
X-100.
4. Incubate in zymogram reaction buffer at 37 °C for specified
time periods (see Note 10).
3.3 Coomassie 1. After incubation, zymogram gel was stained with CBB staining
Brilliant Blue (CBB) solution (see Note 16). Agitate 15–20 min on slow rotary shaker.
Staining 2. Pour out staining solution and add destaining solution
(Fig. 1b) (see Note 17).
a b c
M NJ-2 NJ-3 M NJ-2 NJ-3 M NJ-2 NJ-3
Binding
Protease
(see Note 18)
24 kDa
Fig. 1 SDS-PAGE and fibrin zymography of culture supernatants from two Bacillus sp. strains NJ-2 and NJ-3.
SDS-PAGE was performed and fibrin (0.12%, w/v) zymograms (12% polyacrylamide) were run. After electro-
phoresis, the SDS-PAGE gel was silver-stained (a). Zymograms (b, c) were incubated in enzyme reaction buffer
for 16 h at 37 °C. One gel was stained with CBB (b) and the other gel was silver-stained (c) (reproduced from
[6] with permission from Springer)
3.4 Silver Staining 1. Transfer SDS gel (Fig. 1a) and zymogram gel (Fig. 1c) to a
clean small container (see Note 19).
2. Soak the gel in fixing solution for at least 20 min with gentle
agitation (see Note 20).
3. Wash the gel in 50% methanol for at least 10 with 2–3 changes
(see Note 21).
4. Sensitize the gel in sensitizing solution for only 1 min
(see Note 22).
5. Wash the gel in distilled water for 20 s with three changes
(see Note 23).
6. Incubate the gel in silver staining solution for 20 min
(see Note 11).
7. Wash the gel in distilled water for 20 s with two changes
(see Note 24).
8. Develop the gel in developing solution (see Note 25).
9. Stop development SDS gel (Fig. 1a) and zymogram gel
(Fig. 1c) by rinsing in stop and storage solution.
10. Repeat this procedure with pre-stained protein size markers
(a PageRuler prestained protein ladder) to compare silver-
stained SDS gel (Fig. 2a), CBB-stained common zymogram
gel (Fig. 2b), and silver-stained zymogram gel (Fig. 2c)
(see Note 26).
a b c 170
130
95
*
72
55
43
34
26
17
10
kDa
Fig. 2 Pre-stained protein size markers (a PageRuler pre-stained protein ladder)

on the SDS-PAGE gel (a) and zymograms (b, c) were stained with silver (a, c) or
with CBB (b) (reproduced from [6] with permission from Springer)
4 Notes
1. Two Bacillus sp. strains were grown at 37 °C in a tryptic soy

broth (TSB, Difco, Detroit, USA) for 2 days. The cells were
precipitated by centrifugation at 10,000 × g for 10 min. The
supernatants were used as protease samples.
2. The supernatants (300 μL amounts) were concentrated by
adding 3 volumes of cold acetone (900 μL amounts) for 3 h at
−20 °C. The mixture was centrifuged for 5 min in an Eppendorf
centrifuge. Remove supernatant and air dry pellet. Resuspend
pellet in zymogram sample buffer for zymography.
3. Concentrated HCl (6 M) can be used at first and then it would
be better to use a diluted HCl (1 or 2 M) to avoid a sudden
drop in pH below the required pH.
4. Separating gel solution containing 0.12% (w/v) fibrinogen
and thrombin was centrifuged to remove insoluble impurities
which may be induced when SDS stock solution was mixed.
5. Unpolymerized acrylamide is a neurotoxin and a skin irritant
and care should be exercised to avoid skin contact. Always han-
dle with gloves and mask. Transfer the weighed acrylamide to
the beaker inside the fume hood.
6. We recommend the solution is best to prepare freshly each
time.
7. It is convenient to make a 10× stock solution. Prepare 10×
native buffer (0.25 M Tris, 1.92 M glycine) and 10%
SDS. Weigh 30.3 g Tris and 144 g glycine and add distilled
water to 1 L. To make 1× electrophoresis buffer dilute 100 mL
of 10× native buffer to 990 mL with distilled water and add
10 mL of 10% SDS.
8. Non-reducing sample buffer, do not add reducing agents
(DTT or β-mercaptoethanol). Store for weeks at 4 °C and for
months at −20 °C. Zymogram sample buffer needs to be pre-
warmed prior to use (SDS precipitates at 4 °C).
9. After electrophoresis, detergent SDS is removed from the
zymogram gel by washing in 2.5% Triton X-100. This allows
the protease to renature and the substrate is degraded during
incubation in the zymogram reaction buffer.
10. In order to detect proteolytic activity, it is necessary to work
the protease in a pH of reaction buffer. Therefore, it is best to
check the optimal pH for activity of your protease prior to
applying zymography and then the buffer needs to be changed
if necessary (optimal pH-conditioned buffer).
11. We recommend the solution is best to prepare freshly each time
(add formaldehyde just before use). Vapors of formaldehyde are
very irritating. Thus, store well-closed at room temperature

(according to the manufacturer’s datasheet).
12. The separating gel will fill approximately 80–85% of volume
between the glass plates. Isobutanol or distilled water keeps
the separating gel surface flat. Gels can be stored for up to a
week at 4 °C wrapped in wet plastic wrap.
13. Polymerization of the separating gel is completed when a dis-
tinct layer is formed between the upper edge of the separating
gel and isobutanol or distilled water.
14. Centrifuging the samples prior to preparation for loading helps
remove insoluble material which may interfere with electro-
phoresis [12]. Filling the unused wells with 1× sample buffer is
good for avoiding edge effects. When using unknown samples
to the first attempt to zymography we recommend non-dena-
turing systems. Some proteases are inactive under reducing
condition and many proteases denatured by heat treatment.
15. It is advisable to cool the gel during electrophoresis to keep
the inactive state of proteases in sample and maintain substrate
in the gel not to be degraded during the electrophoresis by the
proteases.
16. Use gloves to prevent staining hands. Cover container with lid
during staining and destaining.
17. Activity in zymograms is visualized as clear bands or binding
mode (see Note 18) against blue background, where active
proteases have degraded the fibrin substrate.
18. We found two types of binding modes. One was formed
because of binding between the enzyme and substrate [7, 8]
and the other was due to a high pI value (8.8) of the enzyme
[13]. These two types were identified using reverse zymogra-
phy and diagonal electrophoretic zymography.
19. Wear gloves at all times when handling the gel and use a clean
(acid-washed) staining tray with lid to avoid contamination of
the gel. Agitate the gel and carry out in a fume hood. An alter-
native silver staining methods or commercial silver staining kits
[ProteoSilver Plus (Sigma) or Dodeca Silver Stain (Bio-Rad)]
are available.
20. Gel may be stored overnight at this step.
21. Washing with 2–3 changes will remove acetic acid in fixing
solution, reduce background staining and increase sensitivity.
22. Longer time will decrease small protein (peptide) recovery
from the gel.
23. Make sure gel is completely immersed. Sensitizer should be
completely removed. Residual sensitizer increase dark uniform
background.
24. Transfer the gel in a new container. Residual silver nitrate on

the gel and staining container will increase background
staining.
25. Change developing solution immediately when it turns yellow.
Stop the step when the staining is sufficient.
26. We recommend Pre-stained size marker in zymogram gel,
which can be seen before and after staining. Plus, it is possible
to determine the band against the dark background after silver
staining.
Acknowledgements
This research was supported by the Basic Science Research Program

through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education (grant number
2013R1A1A2062475).
References
1. Gupta R, Beg QK, Lorenz P (2002) Bacterial 8. Lantz MS, Ciborowski P (1994) Zymographic
alkaline proteases: molecular approaches and techniques for detection and characterization
industrial applications. Appl Microbiol of microbial proteases. Methods Enzymol
Biotechnol 59:15–32 235:563–594
2. Godfrey T, West S (1996) Introduction to 9. Laemmli UK (1970) Cleavage of structural
industrial enzymology. In: Godfrey T, West S protein during the assembly of the head of bac-
(eds) Industrial enzymology, 2nd edn. teriophage T4. Nature 227:680–685
Macmillan, London, pp 1–8 10. Choi NS, Kim BH, Park CS et al (2009)

3. Kim SH, Choi NS, Lee WY (1998) Fibrin Multiple-layer substrate zymography for
zymography: a direct analysis of fibrinolytic detection of several enzymes in a single

enzymes on gels. Anal Biochem 263:115–116 sodium dodecyl sulfate gel. Anal Biochem
4. Kim SH, Choi NS (1999) Electrophoretic 386:121–122
analysis of protease inhibitors in fibrin zymog- 11. Choi NS, Choi JH, Han YJ et al (2009) Mixed-
raphy. Anal Biochem 270:179–181 substrate (glycerol tributyrate and fibrin)
5. Choi NS, Kim SH (2000) Two fibrin zymogra- zymography for simultaneous detection of
phy methods for analysis of plasminogen acti- lipolytic and proteolytic enzymes on a single
vators on gels. Anal Biochem 281:236–238 gel. Electrophoresis 30:2234–2237
6. Chung DM, Kim KE, Ahn KH et al (2011) Silver- 12. Hames BD (1981) Gel electrophoresis of

stained fibrin zymography: separation of proteases proteins. In: Hames BD, Rickwood D (eds)
and activity detection using a single substrate-con- A practical approach. IRL Press, London,
taining gel. Biotechnol Lett 33:1663–1666 p 290
7. Brown TL, Yet MG, Wold F (1982) Substrate- 13. Park CS, Kang DO, Lee WY et al (2015)
containing gel electrophoresis: sensitive detec- Identification of two types binding modes
tion of amylolytic, nucleolytic, and proteolytic using reverse or diagonal electrophoretic
enzymes. Anal Biochem 122:164–172 zymography. Acad J Biotechnol 3:52–55
Chapter 17
Zymography Methods to Simultaneously Analyze

Superoxide Dismutase and Catalase Activities: Novel
Application for Yeast Species Identification
Esther Gamero-Sandemetrio, Rocío Gómez-Pastor, and Emilia Matallana
Abstract
We provide an optimized protocol for a double staining technique to analyze superoxide dismutase enzy-
matic isoforms Cu-Zn SOD (Sod1) and Mn-SOD (Sod2) and catalase in the same polyacrylamide gel. The
use of NaCN, which specifically inhibits yeast Sod1 isoform, allows the analysis of Sod2 isoform while the
use of H2O2 allows the analysis of catalase. The identification of a different zymography profiling of SOD
and catalase isoforms in different yeast species allowed us to propose this technique as a novel yeast iden-
tification and classification strategy.
Key words SOD, Catalase, Zymogram, Electrophoretic isoforms, Yeast
1 Introduction
Separation of enzymes by polyacrylamide gel electrophoresis

(PAGE) followed by zymography is a widely used technique for
identifying enzymatic activities [1]. The term zymogram refers
to the analysis of an enzymatic activity [2] and the term zymog-
raphy (introduced in 1962, by Gross and Lapière [3]) involves
the electrophoretic technique for the detection of a particular
enzyme, based on the substrate repertoire for such enzyme.
With zymography, the enzyme substrate is converted into a
product that can literally be visualized. The biochemical reac-
tion is measured by detection methods that analyze either
appearance of the reaction product or disappearance of the sub-
strate [4]. This technique provides reliable identification of
enzymes based on the electrophoretic mobility of their active
forms after gel electrophoresis.
The conventional zymography technique was created for the
identification of enzymes with protease activity and it was based on
a sodium dodecyl sulfate gel impregnated with a protein substrate,
189
190 Esther Gamero-Sandemetrio et al.
which is degraded by the proteases resolved during the incubation

period. Coomassie blue staining of the gel reveals sites of proteoly-
sis as white bands on a dark blue background [1]. Over the last 4
decades, zymography has been transformed into different forms,
such as chromogenic substrate autography (substrate indicator
gel), reverse zymography, 2D-zymography, in situ zymography,
in vivo zymography [5] and native PAGE-coupled zymography [6]
allowing to identify not only hundreds of proteases, but also other
enzymes such as lipases, esterases, catalase, and superoxide dis-
mutase (SOD). Moreover, the advantage of this technique is that
any kind of biological sample can be analyzed, including cell and
tissue extracts, whole blood, plasma, and other complex body or
lavage fluids.
The analysis of antioxidant enzymes like SOD and catalase has
important implications in cosmetic, chemical, food, and pharma-
ceutical industries because of their capability to catalyze the
destruction of reactive oxygen species and prevent oxidation.
Currently, the most popular method to detect SOD and catalase
activity is by native PAGE-coupled zymography [7, 8]. This
method has the advantage of detecting only active enzymes across
the spectrum of different types of SOD and catalase and discrimi-
nating between different enzyme isoforms.
SODs are a class of highly conserved enzymes that catalyze
the dismutation of superoxide in oxygen and hydrogen peroxide
[9]. Different SOD isoenzymes are currently classified according
to the metal cofactor in their active site. Therefore, we can clas-
sify SODs into four main types: CuZn-SOD, Mn-SOD, Fe-SOD,
and Ni-SOD. In the yeast Saccharomyces cerevisiae, there are two
main distinct superoxide dismutases: CuZn-SOD (Sod1) and
Mn-SOD (Sod2). Sod1 is mainly located in the cytosol although
a small percentage can be found in the intermembrane space of
the mitochondria [10], while Sod2 is located in the mitochon-
dria. In Candida albicans, an irregular cytosolic Mn-SOD (Sod3)
has been reported [11]. In addition, this yeast has other three
additional genes for expressing Sod1-related GPI-anchored
extracellular proteins Sod4, Sod5, and Sod6. Interestingly, it has
been recently showed that Sod5 represents a unique class of
Cu-only SOD, required for pathogen defense [12]. On the other
hand, there are two described catalases, Cta1, which localizes in
the peroxisome, and Ctt1, which is cytosolic. They reduce hydro-
gen peroxide using the redox properties of a heme group com-
plexed to the polypeptide [13].
The two different SOD isoforms (Sod1 and Sod2) of S. cerevi-
siae can be separated and visualized in PAGE zymograms by using
nitro blue tetrazolium (NBT) staining [7, 14], where SOD bands
are visualized as clear bands on a blue background. Inhibition of
Sod1 can be achieved by using NaCN, which allows the specific
Superoxide Dismutase and Catalase Zymography 191
detection of Sod2 isoforms [15]. In the case of catalase isoforms,

they can be separated on starch gel zymograms although their sim-
ilar molecular weights in yeast make it difficult to separate them on
normal PAGE zymograms [16]. However, the activity of Ctt1,
which is vital in protecting yeast against exogenous H2O2, is much
higher than the activity of Cta1 under physiological conditions
[17]. Therefore, the catalase activity observed in a PAGE zymo-
gram can be attributed mainly to Ctt1, without the need to sepa-
rate both isoforms. When a 3,3′-diaminobenzidine (DAB)
peroxidase stain is applied to a polyacrylamide gel, followed by a
ferricyanide negative stain, catalase appears as clear bands on a
green background [18].
Despite the high sequence conservation of SOD and catalase
across the evolution, polymorphic forms of these enzymes have
been reported under different conditions. Interestingly, those
polymorphisms alter the total charge of the protein, affecting their
electrophoretic mobility and creating a unique electrophoretic
profile in different organisms. In human Sod1 mutant alleles carry-
ing amino acidic changes E100G and E100K, the lack of one nega-
tive charge or the increase in total positive charge respectively,
resulted in a diminished Sod1 zymogram mobility compared to
non-mutated SOD [19]. Unique SOD and catalase profiles are
also exhibited by different Deinococcus and Naegleria species and
the zymogram patterns of theses enzymes have been used to dif-
ferentiate these bacteria and ameba [7, 20]. This differentiation
was possible by a double-staining zymogram technique to visualize
SOD and catalase bands in the same gel [7]. However, the poly-
morphic characterization of these enzymes in different yeast spe-
cies has never been explored.
In this chapter, we describe an optimized double staining pro-
tocol for Sod1, Sod2, and catalase zymogram detection to identify
new enzyme polymorphic forms in different yeasts. This strategy
provides a unique SOD-catalase zymogram profiling, which can be
used as a novel approach to discriminate and classify different
Saccharomyces and non-Saccharomyces yeast species.
2 Materials
Prepare all solutions using ultrapure water (obtained by purifying

deionized water to attain a sensitivity of 18 MΩ cm at 25 °C) and
analytical grade reagents.
2.1 Buffers, 1. Extraction buffer: 0.05 M Tris–HCl, pH 8.0. Weight 6.06 g

Solutions, Tris and dissolve in about 900 mL of water. Mix and adjust pH
and Reagents with HCl. Make up to 1 L with water. Store at 4 °C.
2. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Weigh 181.7 g

Tris and prepare 1 L solution. Store at 4 °C.
Tris and prepare 1 L solution. Store at 4 °C.
4. SS4X load buffer: sucrose 20% (m/v), 0.25% bromophenol
blue (m/v).
5. Running buffer: Weight 3.03 g Tris, 14.41 g glycine. Add
water to a volume of 500 mL. Mix and adjust pH to 8.0 with
HCl. Make up to 1 L with water. Store at 4 °C.
6. NaCN Inhibitor solution: 1 mM NaCN. Weigh 25 mg NaCN
and mix in 500 mL water. Store at 4 °C.
7. SOD staining solution: 80 mL of 0.05 M Tris–HCl pH 8.0
containing 10 mg MTT [3-(4,5(dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide], 6 mg PMS (phenazine metho-
sulfate) and 15 mg MgCl2 (see Note 1).
8. H2O2 solution: 100 mL of 50 mM phosphate buffer (pH 7.8)
containing 0.04 M H2O2.
9. Catalase staining solution: 20 mL 2% (m/v) ferric chloride and
20 mL 2% (m/v) potassium ferric cyanide in water (see Note 2).
10. Acrylamide/Bis-acrylamide (29:1), 30% solution.
11. Bradford Reagent.
12. Bovine serum albumin (BSA) stock 1 mg/mL.
13. Ammonium persulfate: 10% solution in water.
14. N,N,N′,N′-Tetramethylethylenediamine (TEMED).
2.2 Special 1. FastPrep-24 5G cell homogenizer.

Equipment 2. Protein electrophoresis equipment: XCell SureLock™ Mini-
Cell Electrophoresis System.
3. Image analyzer: Luminescent Image Analyzer LAS-1000plus
and Image Gauge software.
3 Methods
The method described in this section allows the identification of

different polymorphic isoforms for Sod1, Sod2 and catalase in dif-
ferent yeast species:
1. Collect 50 mg of yeast cell samples for protein extraction by
centrifuging at 2200 × g for 3 min at 4 °C.
2. Wash cells in 50 mL water and centrifuge at 2200 × g for 3 min
at 4 °C.
3. Resuspend cells in 500 μL extraction buffer containing the

mixture of protease inhibitors 1× (Roche, Switzerland) and
0.4 g of glass beads (see Note 3).
4. Cells are lysed in a FastPrep homogenizer by two 30 s breaking
cycles at 5.0 m/s and then centrifuged at 1620 × g for 3 min
at 4 °C.
5. Determine protein concentration in the extracts by using the
Bradford assay and a BSA calibration curve (10–70 μg/mL).
6. All samples are diluted to 20 μg/μL in 0.05 M Tris–HCl
pH 8.0, and resuspended in SS4X load buffer to a final concen-
tration of 1×.
7. 80 μg of total protein of each sample were separated in 10%
native-PAGE (Table 1) developed in running buffer for 1 h at
15 mA and 4 °C. Two gels were performed in parallel, one was
used to analyze Sod1 and Sod2 isoforms (Gel A) and the other
was used to identify Sod2 isoform (Gel B).
8. Gel A was immersed in distillated water and Gel B was
immersed in 1 mM NaCN, which specifically inhibits Sod1p
isoform (see Note 4). Both gels were incubated in their corre-
sponding solutions for 4 min at approximately 20 °C.
9. Immediately, both gels were immersed in 80 mL of SOD stain-
ing solution.
10. The gels were exposed to sunlight for 5–10 min to visualize
the SOD bands as white bands on a blue-black background (see
Note 5).
Table 1
Composition of 10% native acrylamide gel
Stacking gel
Tris–HCl (pH 6.8) 125 mM
Acrylamide/Bis-acrylamide (29:1) (m/m) 5% (v/v)
Ammonium persulfate 0.05% (m/v)
TEMED 0.025% (v/v)
Resolving gel
Tris–HCl (pH 8.8) 375 mM
Acrylamide/Bis-acrylamide (30:0.8) (m/m) 10% (v/v)
Ammonium persulfate 0.05% (m/v)
TEMED 0.025% (v/v)
11. Gel A (corresponding to Sod1 and Sod2 isoforms) and Gel B

(corresponding to Sod2 isoforms) images are captured with
Luminescent Image Analyzer LAS-1000plus Camera for fur-
ther analysis (see Note 6).
12. Afterward, Gel A is washed with distilled water for 5 min and
immersed in 100 mL of 50 mM phosphate buffer (pH 7.8)
containing 0.04 M H2O2 for 20 min at room temperature in
darkness and shaking (Gel A + H2O2).
13. Then, the gel is washed with distilled water for 5 min and
immersed in catalase staining solution, shaking it under sun-
light for 5 min to visualize the yellow bands of catalase.
14. Stop reaction by transferring the gel into distilled water.
15. Gel image acquisition was performed in a Luminescent Image
Analyzer LAS-1000plus Camera.
16. Finally, the combined analysis of the three different captured
images Gel A (Sod1, Sod2), Gel B (Sod2), and Gel A + H2O2
(Catalase) shows a specific zymographic profiling for every
yeast species (see Figs. 1, 2, and Table 2). See also Ref. 16 for
published results.
Figure 1 shows a schematic description of the SOD-catalase
double staining technique for the identification of specific yeast
polymorphic isoforms and an example of previously published
results is shown in Fig. 2 and Table 1 [19].
4 Notes
1. Weigh each reagent in separate tubes and dissolve them in

1 mL 0.05 M Tris–HCl, pH 8.0. MTT resuspension is difficult
so it is recommended to cut the extreme of the tip. Keep PMS
and MTT in the darkness prior to prepare the final solution.
Add the reagents to the final volume solution in the following
order: MgCl2, PMS, and MTT.
2. Prepare 2% (m/v) ferric chloride and 2% (m/v) potassium fer-
ric cyanide solutions separately and mix them into the final
catalase solution right before use.
3. Add 1 tablet of protease inhibitor to 50 mL 0.05 M Tris–HCl,
pH 8.0, at the time of use.
4. NaCN inhibits specifically Sod1 allowing the identification of
bands belonging to Sod2. The combined analysis of treated
and untreated gels allows the identifications of the different
Sod bands in each yeast species. We have also used another
specific inhibitor, diethyldiothiocarbamate (DDC) but it does
not efficiently inhibit Sod1 in zymogram analysis.
Fig. 1 Diagram of the catalase and SOD double staining zymogram technique used for the resolution of different
electrophoretic isoforms in several yeast species. In the zymogram profile of superoxide dismutases Sod1 and
Sod2, the different electrophoretic Sod1 isoforms (a and b) are indicated with white asterisks and the unique Sod2
isoforms is indicated as (a′). In the zymogram profile of catalase the different isoforms are labeled as a, b, and c.
The inserted table shows the zymogram profile of SOD and catalase for the hypothetical yeast strains
Fig. 2 Identification of SOD and catalase isoforms from Saccharomyces and non-Saccharomyces species. (A)
Zymogram profile of superoxide dismutase Sod1 and Sod2 for different Saccharomyces yeast strains after
24 h of growth in molasses. (B) Zymogram profile after soaking an identical acrylamide gel in NaCN to specifi-
cally inhibit the Sod1 isoform. (C) Zymogram profile of Sod1 and Sod2 for different non-Saccharomyces wine
yeast strains cultured in molasses medium. (D) Zymogram profile after soaking an identical acrylamide gel in
NaCN. In all the panels, Sod1 isoforms (a–f) are indicated with white triangles and the different electrophoretic
isoforms of Sod2 (a′–b′) are indicated with white spots. (E) Catalase zymogram profile for yeast belonging to
the genus Saccharomyces. (F) Catalase zymogram profile for other oenological non-Saccharomyces yeast
species. Catalase (a), (b), and (c) correspond to different electrophoretic isoforms
5. Although the Sod zymogram can be developed with a white

light lamp, the use of sunlight provides faster developing and
better bands resolution.
6. These gels can also be used for quantification of the SOD
activity. For this, the gel has to be stained with Coomassie blue
to calculate the total loaded protein (Pi) and this value is used
to normalize the signal data (Ci/Pi). In parallel, Sod1p and
Sod2p western blots allow to calculate the specific activity
[(Ci/Pi)/Sodp].
Table 2
SOD and catalase zymographic isoforms
Catalase
Strain Sod1p isoform Sod2p isoform isoform
S. cerevisiae (S.c) a a a
S. bayanus (var. uvarum) b a b
(S.u)
S.c x S.u b a b
S.c x S.b (var. bayanus) – a a
S. paradoxus (S.p) b a c
S. kudriavzevii (S.k) d – –
S.c x S.k a,c a a
Candida stellata – a b
Candida albicans c b b
Torulaspora delbruekii – b b
Pichia fermentans e – b
Pichia pastoris e – a
Hanseniospora osmophila b,f – –
Hanseniospora d – –
guilliermondii
Acknowledgements
This work has been supported by grants AGL2011-24353 and

AGL2014-52985-R from the Spanish Ministry of Economy and
Competitiveness (MINECO) to E.M., and it has been performed
within the Program VLC/Campus, Microcluster IViSoCa
(Innovation for a Sustainable Viticulture and Quality), and
Microcluster BBLM (Model Yeasts in Biomedicine &
Biotechnology). E.G.-S. was a predoctoral fellow of the JAE pro-
gram from the CSIC (Spanish National Research Council). R.G.-P.
was a postdoctoral researcher at Universitat de València.
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10. Sturtz LA, Diekert K, Jensen LT, Lill R, 18. Gamero-Sandemetrio E, Gómez-Pastor R,
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tive damage. J Biol Chem 276:38084–38089 Biotechnol 97:4563–4576
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FZ (2014) Superoxide dismutase 1 acts as a Subramaniam JR, Wong PC, Valentine JS,
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Chapter 18
Detection of Guaiacol Peroxidase on Electrophoretic Gels

Diana Castro, Lellys M. Contreras, Liliana Kurz, and Jeff Wilkesman
Abstract
It is possible to analyze peroxidase (POD) from different vegetable sources by electrophoresis. Zymography,
i.e., a SDS-PAGE method to detect enzyme activity, is used to specifically detect POD activity and to
visualize the total protein profile. For this purpose, we describe how a radish homogenate is prepared
and submitted first to electrophoresis, and then, the POD activity present in the gel is reactivated and
selectively stained using guaiacol as substrate. After scanning the gel, the same gel is further stained with
Coomassie blue to determine the whole protein profile of the sample.
Key words Guaiacol peroxidase, Electrophoresis, Zymography
1 Introduction
The large family of enzymes named peroxidases (EC 1.11.1.x),

typically catalyze the reaction type ROH+HOR′:
ROOR¢ + 2e- + 2H + ¾¾¾¾ ® ROH + HOR¢
Peroxidase
Many of these enzymes use hydrogen peroxide as substrate, yet
others are specific towards organic hydroperoxides such as lipid per-
oxides. Peroxidases may contain a heme cofactor in their active sites,
or alternately, redox-active cysteine or selenocysteine residues.
POD from radish (Raphanus sativus) belongs to the ferropro-
toporphyrin group of peroxidases. It has been described as a gly-
coprotein conformed by two polypeptide chains, having a Mr of
31,965.5 each, containing four disulfide bridges with an average
Mr of 66,000 Da. POD catalyzes H2O2 decomposition, yet the
resultant [HRP–H2O2] intermediate complex can oxidize a variety
of chromogenic hydrogen donors. The use of chemiluminescent
substrates such as luminol or isoluminol, and fluorogenic substrates
such as 4-hydroxyphenyl acetic acid, homovanillic acid, and tyra-
mine, is also possible. Though many different kinds of c hromogenic
substrates for peroxidases are reported (o-dianisidine, benzidine,
199
200 Diana Castro et al.
diaminobenzidine, etc.), the procedure proposed employs guaiacol,

a non-carcinogenic substrate [1]. Visualization of the enzyme
activity is performed after a reddish product is produced in minutes,
and the molecular mass, subunits conformation, enzymatic activity,
and densitometry can be conveniently further analyzed.
The detection of enzyme activity on electrophoretic gels, or
zymography, has become an important tool to evaluate and ana-
lyze many enzymes [1–3]. Herein, we describe the analysis of
radish peroxidase (POD) using as substrate guaiacol, employing
a safe, economical, and effective zymography experiment. With
this technique, enzymatic activity is visualized as reddish bands
on a translucent gel. Afterwards, the total protein content may be
visualized by staining with Coomassie blue.
2 Materials
2.1 Solutions for Gel 1. Acrylamide: bis-acrylamide 30:0.8: Stock solution is prepared

Preparation by weighing 30 g acrylamide and 0.8 g bis-acrylamide. Dissolve
in 100 mL water and filter. Solution is stable at 4 °C for
months (see Note 1).
2. 10% (w/v) APS: weigh 0.1 g APS and dissolve in 1 mL
(see Note 2).
3. 1.5 M Tris–HCl buffer pH 8.8: Prepare 100 mL of solution
(see Note 3).
4. 1 M Tris buffer pH 6.8: Prepare 100 mL of solution (see Note 3).
2.2 Buffers 1. Sample buffer 4×: 200 mM Tris pH 6.8, 4% (w/v) SDS, 40%
and Solutions (v/v) glycerol, 0.02% (w/v) bromophenol blue.
2. Running buffer 10×: 250 mM Tris–HCl pH 8, 0.1% (w/v)
SDS, 192 mM glycine.
3. Reactivation solution: 1% (v/v) Triton X-100 (see Note 4).
4. Solution A: pipette 10 μL 25% (v/v) guaiacol to 1.5 mL ethanol,
and complete final volume to 10 mL with 50 mM phosphate
buffer pH 7.
5. POD staining solution: Mix 10 mL of solution A with 9 mL
50 mM phosphate buffer pH 7 and add 1 mL 0.3% (v/v)
hydrogen peroxide (see Note 5).
6. Coomassie staining solution: 0.018 mg/mL Coomassie blue
R-250, 1% (v/v) acetic acid, 24% (v/v) methanol (see Note 6).
7. Destaining solution: 1% (v/v) acetic acid, 24% (v/v) methanol.
Guaiacol POD Zymography 201
3 Methods
3.1 POD Extraction 1. Rinse and cut one or two pieces of fresh radish (or any other
vegetable source to be tested) into small pieces.
2. Weigh ~25 g of the pieces and place them in a blender with
a minimal amount (~20 mL) of cold 50 mM phosphate buffer
pH 7.
3. Filter the resulting mixture through cheesecloth, so that the big
particles are retained and only the liquid extract (homogenate)
is obtained.
4. Spin briefly. Aliquot 1 mL of the homogenate and keep on ice
until electrophoresis (see Note 7).
5. If the effect of concentration is to be analyzed, aliquot different
quantities of homogenate (i.e., 50, 25, 10, 5, 1 μL) into sample
buffer 4× (see Note 8).
3.2 Electrophoresis 1. Choose small gels (8 × 10 cm2) as they run faster and clean
glasses thoroughly (see Note 9).
2. Prepare the resolving and stacking gels according to Table 1
3. Run gels at 200 V (~ 40 mA for two mini-gels) at 4 °C.
Run until the blue front reaches the bottom of the gel (see
Notes 12 and 13).
Table 1
Preparation of SDS-PAGE mini-gels (8 × 6 cm2)
Volumea/(mL)
Stock solution Stock Resolution gel Stacking gel

component concentration (10%) (4.5%)
H2O (d) – 3.28 2.2
Acrylamide mix 30% 2.67 0.6
Tris-HCl pH 8.8 1.5 M 2.00 –
Tris-HCl pH 6.8 1 M – 1.0
APSb 10% 0.08 0.03
TEMEDc 100% 0.008 0.001
Final volume: ~8 ~4
a
Volume given for two mini-gels, 4 mL each
b
Ammonium persulfate
c
N,N,N′,N′ Tetramethylethylenediamine
3.3 Gel Staining 1. Immediately after the run, place the gel in a Petri dish, previously
filled with reactivation solution.
2. Incubate twice with gentle shaking for 15 min.
3. Discard reactivation solution and add POD staining solution.
Shake gently until reddish bands in a clear background appear
(see Note 14).
4. After staining, gel is scanned or photographed, since color fades
with time, and also because after the next staining step, the speci-
ficity for peroxidase-stained bands will be lost (see Notes 15
and 16).
5. Discard the solution in the Petri dish. Add Coomassie solution
to the gel. And leave with gentle shaking, typically overnight
(see Note 17).
6. Discard the staining solution (see Note 18), and remove the
excess of dye from the gel with destaining solution (see Note 19).
Leave shaking gently.
7. Stop destaining once blue bands with a clear or bluish back-
ground are obtained.
8. Gels can be now scanned, photographed, or dried (see Note 20).
Figure 1 presents a typical result. According to Wilkesman
et al. [4], the sensitivity limit of the assay is around 4 μg of total
Fig. 1 Analysis of radish POD. (a) 10% Zymogram. (b) 10% SDS-PAGE stained with
Coomassie blue. Lanes: (1) 4.4 μg total protein; (M) Molecular weight marker (in kDa):
(1) Myosin (229), (2) phosphorylase (116), (3) BSA (82.2), (4) ovalbumine (49.1),
(5) Carbonic anhydrase (32), (6) Trypsin inhibitor (25.7), and (7) Lysozyme (17.7)
Guaiacol POD Zymography 203
protein per lane, in order to visualize efficiently POD activity.

Figure 1a shows the zymogram with POD activity and Fig. 1b
shows the protein profile stained with Coomassie blue.
4 Notes
1. Acrylamide is a proven neurotoxin, with carcinogenic and

mutagenic effects; extreme care must be taken. Wear gloves
when handling Acrylamide powder and solutions. Wear a safety
mask when weighing the acrylamide solid. Another option is to
buy the already made solution. In this case, it is just necessary to
aliquot the liquid in small portions for better handling.
2. Prepare solution fresh. Normally 1 mL is prepared in a micro
vial. Solution is stable for 2 weeks at 4 °C.
3. If stored at 4 °C, check pH before use.
4. Normally, 50 mL/gel is enough. As it will be used twice, prepa-
ration of 100 mL is enough for one gel.
5. Some guaiacol preparations are quite smelly. Maintain guaiacol
solutions in the fume hood. Normally, 50 mL is enough to
cover the gel placed in a Petri dish.
6. Methanol is a volatile, flammable, and toxic organic solvent.
7. For POD detection, radish homogenate must always be fresh.
Refrigeration of the samples does not assure POD activity
throughout time. Additionally, a parallel and complementary
enzymatic activity test is suggested to be performed, e.g., pho-
tometry test at 470 nm [5–7].
8. It is strongly recommended to measure the total protein con-
centration of the homogenate by some of the traditional
methods reported in the literature (Bradford, BCA, Lowry,
UV, etc.) [8–11]. In this manner, it is possible to determine
the amount (μg) of proteins introduced in each lane of the gel.
Moreover, it is advised to reserve a lane for a commercial
peroxidase to be used as a positive control.
9. Wear gloves when manipulating the glasses.
10. To optimize time, during gel polymerization, the POD extract
can be prepared.
11. Commercial precasted gels may be used as well.
12. The amperage values used during electrophoresis are poten-
tially lethal. During the run, the electrophoretic unit must not
be touched.
13. Typical running times are between 35 and 50 min.
14. Bands should be visualized in the next 5 min. However, time
for band apparition varies from 30 s until 10 min. Longer
incubation times normally suggest enzyme inactivation.
15. Record (scan, photograph) the results immediately after band

apparition as bands do fade away with time.
16. Densitometry analysis may be performed at any time later, as long
as the file containing the gel image has been properly saved.
17. Staining times may vary from a couple of hours until over-
night. Other Coomassie recipes may be used.
18. Coomassie staining solution may be reused several times. Have
ready an empty glass bottle in order to recover the solution
after staining the gels.
19. Destaining solution may be recovered too in an amber glass
bottle filled with some active charcoal in the bottom. After
1 month, the solution is clear and may be reused.
20. If the polyacrylamide gel is to be discarded, do it appropriately.
Acknowledgments
We thank the Consejo de Desarrollo Científico y Humanístico,

Universidad de Carabobo and LOCTI for partial funding of this
work (CDCH-UC-1080-07).
References
1. Manchenko G (2003) Handbook of detection plasma membranes. Plant Physiol 132:

of enzymes on electrophoretic gels, 2nd edn. 1489–1498
CRC Press LLC, USA, pp 160–162 7. Doerge DR, Divi RL, Churchwell MI (1997)
2. Wilkesman J, Kurz L (2009) Protease analysis Identification of the colored guaiacol oxidation
by zymography: a review on techniques and product produced by peroxidases. Anal
patents. Recent Pat Biotechnol 3:175–184 Biochem 250:10–17
3. Wilkesman J, Kurz L (2012) Advances in zymog- 8. Westermeier R (1997) Electrophoresis in prac-
raphy techniques and patents regarding protease tice, 2nd edn. Wiley-VCH, Weinheim, pp 5–36.
analysis. Recent Pat Biotechnol 6:106–114 165–186
4. Wilkesman J, Castro D, Contreras LM, Kurz L 9. Mikkelsen S, Cortón E (2004) Bioanalytical
(2014) Guaiacol peroxidase zymography for chemistry. John Wiley and Sons, USA,
the undergraduate laboratory. Biochem Mol pp 167–184
Biol Educ 42:420–426 10. Rosenberg IM (2005) Protein analysis and
5. Ghamsari L, Ezzatollah K, Shokoofeh G purification, 2nd edn. Birkhäuser, Boston,
(2007) Kinetics properties of guaiacol peroxi- pp 63–79
dase activity in Crocus sativus L. corm during 11. Walker JM (2009) The protein protocols hand-
rooting. Iran Biomed J 11:137–146 book, 3rd edn. Humana Press, New York,
6. Mika A, Lüthje S (2003) Properties of guaiacol pp 177–186
peroxidase activities isolated from corn root
Chapter 19
In Situ Demonstration and Characteristic Analysis

of the Protease Using Substrate Immersing Zymography
HaiLun He, Hao Li, and Dan Liu
Abstract
Zymography, the detection of proteolytic activities on the basis of protein substrate degradation, has been
a technique described in the literature for at least in the past 50 years. In this study, we used substrate
immersing zymography to analyze proteolysis of proteases. Instead of being directly added into a sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) gel, the substrates were added into the
immersing solution after electrophoresis. With substrate immersing zymography, some characters of
proteases, such as enzyme forms, potential proteolytic activity, molecular weights, presence of complexes,
and potentially active enzyme fragments in complex biological samples, can be determined.
Key words Zymography, Substrate immersing zymography, Protease, SDS–PAGE
1 Introduction
Zymography is known as an electrophoretic technique for the study

of proteolytic activities on the basis of substrate degradation [1].
Zymography was originally established for detecting collagen deg-
radation and describing a matrix metalloproteinase (MMP) in 1962
by Gross and Lapière [2]. In the traditional zymography, proteins
are separated by nonreducing sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) in which the acrylamide is copoly-
merized with a protein substrate. After the electrophoretic run, the
SDS is soaked out from the gel by incubation in a non-buffered
Triton X-100, followed by incubation under conditions that are
conducive for protease activity before staining. The zymogram is
subsequently stained, and light-stained bands contrasted with the
dark-stained background would indicate the protease activity for
hydrolyzing the substrate [3].
In recent years, many new techniques have been developed
based on zymography, such as two-dimensional (2-D) zymogra-
phy, real-time zymography, multiple-layer substrate zymography,
and in situ zymography [4–7]. Vandooren and his colleagues
205
206 HaiLun He et al.
compared different types of zymography experiments and listed

the advantages, disadvantages, and limitations of different zymog-
raphy methods [8]. With the zymography technique, numerous
kinds of biological samples, including cells and tissue extracts,
secretion of cells or tissues, whole blood, plasma, serum, and even
whole organisms can be analyzed [9, 10]. Zymography strategy
has been mainly used to detect potential proteolytic activity or to
analyze the number of active components in a multi-enzyme
system. Furthermore, zymography can be used to analyze the
molecular weights of proteases. Unfortunately, the conventional
zymography often determines molecular sizes of some proteases
inaccurately, due to decreased mobility of proteins in an acrylamide
gel copolymerized with protein substrate [11]. Moreover, the
protein transfer of traditional zymography was of low sensitivity
due to the loss of the protein during the transfer process [12].
In order to overcome these disadvantages of traditional zymog-
raphy, a substrate immersing zymography strategy, which allows
accurate detection of activity and molecular weight of the protease,
was developed in our laboratory. Of course, in the substrate
immersing zymography there is no need to transfer the enzyme to
a substrate gel; therefore, the protease lost is avoided. In this study, a
substrate immersing zymography strategy was performed to detect
proteolytic activity. Moreover, the molecular weight of protease,
the trend of protease production, and components of multi-protease
system of marine bacteria were also analyzed by the modified
zymography method. This technique will provide a useful and rapid
method for detecting potential hydrolytic activities, enzymatic
characteristics, and bacterial multi-protease system.
2 Materials
All solutions were prepared using ultrapure water (prepared by

purifying deionized water to attain a sensitivity of 18.25 MΩ cm at
25 °C) and analytical grade reagents. All reagents were prepared
and stored at room temperature (unless indicated otherwise).
Waste disposal instructions were strictly performed while disposing
waste materials.
2.1 SDS 1. 2 M Tris–HCl, pH 8.8, 100 mL. Weigh 24.2 g Tris and transfer
Polyacrylamide Gel to the cylinder. Add water to a volume of 50 mL (see Note 1).
Mix and adjust pH with HCl (see Note 2). Make up to 100 mL
2.1.1 Stock Solutions
with water. Store at 4 °C.
2. 1 M Tris–HCl, pH 6.8, 100 mL. Weigh 12.1 g Tris and prepare
a 100 mL solution as described in previous step. Store at 4 °C.
3. 10% (w/v) SDS, 100 mL. Weigh 10 g SDS and make up to
100 mL with water. Store at room temperature (~25 °C).
Immersing Zymography of Proteases 207
4. 50% (v/v) glycerol, 100 mL. Measure 50 mL 100% glycerol and

make up to 100 mL with water. Store at room temperature
(~25 °C).
5. 1% (w/v) bromophenol blue (BPB), 10 mL. Weigh 100 mg
BPB and add water to 10 mL, stir until dissolved.
2.1.2 Working Solutions 1. Solution A: 30% acrylamide/Bis solution (29.2:0.8 acrylamide:

Bis): Weigh 29.2 g of acrylamide monomer and 0.8 g Bis
(cross-linker) and transfer to a 100 mL graduated cylinder con-
taining about 50 mL of water, slowly stir until the acrylamide
was completely dissolved. Make up to 100 mL with water and
filter through filter paper (see Note 3). Store at 4 °C, in a bottle
wrapped with aluminum foil (see Note 4).
2. Solution B: 4× Resolving gel buffer, 100 mL. 75 mL 2 M
Tris–HCl (pH 8.8); 4 mL 10% SDS; Add about 21 mL water;
store at 4 °C.
3. Solution C: 4× Stacking gel buffer, 100 mL. 50 mL 1 M
Tris–HCl (pH 6.8); 4 mL 10% SDS; Add about 46 mL water;
store at 4 °C.
4. Ammonium persulfate (APS): 10% (w/v) solution in water,
Store at 4 °C (see Note 5).
5. N,N,N′,N′-Tetramethyl-ethylenediamine (TEMED). Store at
4 °C and wrapped with aluminum foil.
6. 10× SDS-PAGE running buffer, 1 L. Weigh 30 g Tris, 144 g
glycine, 10 g SDS, add water to 1 L, pH 8.3. Store at room
temperature (see Note 6).
7. 5× Loading buffer, 10 mL. 0.6 mL 1 M Tris–HCl (pH 6.8);
5 mL 50% glycerol; 2 mL 10% SDS; 1 mL 1% bromophenol
blue (BPB). Add about 1.4 mL water. Leave one aliquot at
4 °C for current use and store remaining aliquots at −20 °C
(see Note 7).
2.2 Substrate 1. Triton X-100: 2.5% (v/v) solution in 50 mM Tris–HCl buffer,
Immersing pH 7.5. Store at 4 °C (see Note 8).
Zymography 2. 0.2% Gelatin (w/v): dissolved in 50 mM Tris–HCl buffer,
pH 7.5.
3. 0.2% Casein (w/v): dissolved in 50 mM Tris–HCl buffer,
pH 7.5 (see Note 9).
4. Coomassie blue staining solution, 1 L. Mix 1.5 g of Coomassie
brilliant blue R-250, 350 mL methanol, 100 mL acetic acid,
and 550 mL water. Mix thoroughly and store solution at room
temperature.
5. Coomassie blue destaining solution, 1 L. Mix 250 mL methanol,
100 mL acetic acid, and 650 mL water. Mix thoroughly and
store solution at room temperature (see Note 10).
3 Methods

specified.
3.1 Sodium Dodecyl 1. Prepare the resolving monomer solution by combining all
Sulfate- reagents as specified in Table 1 (see Note 11).
Polyacrylamide Gel 2. Pour the solution between the glass plates. Continue to pour
until 2 cm below the top of the short plate is reached. Gently
overlay the gel with isobutanol or water.
3. Vacuum the water out. Prepare the stacking monomer solu-
tion by combining all reagents except the APS and the TEMED
(Table 2). Vortex for 1 min. Total volume 4 mL.
4. Pour the solution between the glass plates. Continue to pour
until the top of the short plate is reached. Insert a 10-well gel
comb immediately without introducing air bubbles.
5. Gently remove the comb and rinse the wells thoroughly with
distilled water or running buffer.
Table 1
Amounts for the preparation of separating gel
Separating gel 20% 15% 12% 10% 7.5%

Solution A/mL 6.7 5 4 3.3 2.5
Solution B/mL 2.5
ddH2O/mL 0.8 2.5 3.5 4.2 5
10%AP/μL 50
TEMED/μL 5 5 5 5 10
Total/mL 10
Table 2
Amounts for the preparation of stacking gel
Stacking gel 5%
Solution A/mL 0.67
Solution C/mL 1.0
10% AP/μL 30
TEMED/μL 5
ddH2O 2.3
Total 4 mL
3.2 Sample 1. Assemble the gel cassette. Fill the assembly with buffer to just
Preparing under the edge of the outer gel plate.
and Electrophoresis 2. Protein samples with proper dilution or concentration are
mixed with the loading buffer at the ratio of 4:1 and placed
directly in the wells (see Note 12).
3. Carry out electrophoresis at 4 °C with constant voltage
(100 V) until the sample has entered the gel and then continue
at 150 V until the bromophenol blue dye reached the bottom
(see Note 13).
3.3 Substrate 1. Following electrophoresis, pry the gel plates open with the use
Immersing of a spatula and then wash the gels three times with Triton
Zymography X-100 (2.5%) for 15 min at room temperature with constant
shaking to remove SDS.
2. Subsequently wash gel three times for 5 min with 50 mM
Tris–HCl buffer.
3. The resolved gel is immersed in a pre-warmed substrate solution
(0.1% casein or gelatin) and kept at 37 °C for 2 h.
3.4 Coomassie Stain 1. After washing, the gels are fixed in 50 mL Bio-Safe Coomassie
stain (or enough to completely cover gel). Gently shake for 3 h.
2. Gels are destained with Coomassie blue destaining solution
for at least 30 min until clear bands indicating proteolytic
activity become visible. Steps are shown in Fig. 1 [13]. Stained
gels can be stored in water (see Note 14).
4 Notes
1. Having water at the bottom of the beaker helps to dissolve

Tris relatively easily. If using a glass beaker, Tris can be dis-
solved faster provided the water is warmed to about 37 °C.
However, the downside is that care should be taken to bring
the solution to room temperature before adjusting pH.
2. Concentrated HCl (approximately 4 mL) can be used at first
to narrow the gap from the starting pH to the required
pH. From then on it would be better to use dilute HCl (1 M)
to avoid a sudden drop in pH below the required pH.
3. Unpolymerized acrylamide is a neurotoxin and care should be
taken to avoid skin contact. Wear disposable gloves when
handling solutions of acrylamide and a mask when weighing
out powder. Polyacrylamide is considered to be nontoxic, but
polyacrylamide gels should also be handled with gloves due to
the possible presence of free acrylamide.
Fig. 1 The process of substrate immersing zymography. In immersing gel zymog-

raphy, samples are separated on a conventional SDS-PAGE gel. Then, the sepa-
rating gel is washed with Triton X-100, and then the gel is immersed in a
substrate solution and incubated for the hydrolytic reaction about 1 h at a certain
temperature. The gel is afterwards stained with Coomassie Brilliant Blue R-250,
followed by destaining
4. A well-sealed acrylamide solution can be stored at 4 °C for up

to 1 month.
5. We find that it is best to prepare this solution fresh each time.
6. Dilute 100 mL of 10× SDS buffer to 900 mL with water. Care
should be taken to add water, since SDS makes bubbles.
7. SDS precipitates at 4 °C. Therefore, the lysis buffer needs to

be warmed prior to use.
8. Triton X-100 would be dissolved faster provided the Tris–HCl
buffer is warmed to about 37 °C. However, the solution
should be taken to 4 °C before using. It is best to prepare this
fresh each time.
9. The buffer temperature is gradually increased with gentle stirring
to 100 °C for about 10 min until a homogenous dispersion is
achieved. The pH is then adjusted if necessary with NaOH and
HCl. Under acid condition, casein is insoluble.
10. The Coomassie Blue destaining solution could be recycled
and reused.
11. Allow space for stacking the gel and gently overlay with isobu-
tanol or water. Add water very, very slowly! Do not mix water
with the resolving gel. This overlay prevents contact with
atmospheric oxygen (which inhibits acrylamide polymerization)
in addition to helping to level the resolving gel solution. The gel
composition must be selected depending on the size of the
protease of interest.
12. Centrifuging the samples prior to the run helps remove insol-
uble debris, which could produce streaks in the protein lanes
(revealed when stained with Coomassie blue). Load samples
slowly to allow them to settle evenly on the bottom of the
well. Be careful not to puncture the bottom of the well with
the syringe needle or pipette.
13. 13 If the current is too large, great heat will denature protein
during electrophoresis, while if the current is too low, protein
bands will disperse due to the extension of electrophoresis
time.
14. Rinsing the gel extensively in water after staining will remove
background and allow proper visualization of the bands.
Acknowledgments
The work was supported by National Natural Science Foundation

of China (31070061, 31370104), Hunan Provincial Natural
Science Foundation of China (13JJ9001), and National Sparking
Plan Project (2013GA770009).
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up of cysteine protease isotypes in the course of Electrophoretic transfer protein zymography.
post-harvest senescence. J Plant Physiol Anal Biochem 411:277–283
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13. Liu D, Yang XH, Huang JF, Wu RB, Wu CL,
7. Hattori S, Fujisaki H, Kiriyama T, Yokoyama He HL, Li H (2015) In situ demonstration
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reverse zymography: a method for detecting ponents from marine bacteria using substrate
activities of matrix metalloproteinases and immersing zymography. Appl Biochem
their inhibitors using FITC-labeled collagen Biotechnol 175:489–501
Chapter 20
Use of Zymography in Trypanosomiasis Studies

Jéssyka Fernanda Santiago Monte, Cláudia Jassica Gonçalves Moreno,
Joana Patrícia Molato Figueiredo Lopes Monteiro, Hugo Alexandre de
Oliveira Rocha, Aline Rimoldi Ribeiro, and Marcelo Sousa Silva
Abstract
Zymography assay is a semiquantitative technique, very sensitive, and commonly used to determine metal
loproteinase levels in different types of biological samples, including tissues, cells, and extracts of protein.
Samples containing metalloproteinases are loaded onto a polyacrylamide gel containing sodium dodecyl
sulphate (SDS) and a specific substrate (gelatin, casein, collagen, etc.). Then proteins are allowed to
migrate under an electric current and the distance of migration is inversely correlated with the molecular
weight. After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary
structure, necessary for enzymatic activity (metalloproteinase activity). In the context of infections caused
by trypanosomatids (Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei), the characterization of
metalloproteinase by zymography can contribute to the comprehension of the pathogenesis mechanisms
and host–parasite interaction.
Key words Zymography, Metalloproteinases, Trypanosomatids, Trypanosoma cruzi, Trypanosoma

brucei, Leishmania spp.
1 Introduction
Zymography is a technique that has been widely used for identifica

tion and characterization of proteases, especially metalloproteinases.
It consists of a proteolytical assay capable of detecting latent and
active forms of enzymes in cells, tissues, biological fluids, and prepa
rations (e.g., cell culture supernatants, crude protein extract, serum,
etc.) [1]. Described since 1980 by Heussen and Dowdle [2], this
technique provides a sensitive, quantifiable, and functional analysis of
metalloproteinases, by gel electrophoresis under denaturing and
nonreducing conditions, being possible to estimate the proteolytic
activity of these proteases based on the degradation of a specific sub
strate, as well as their identification based on molecular weight [3].
Briefly, this technique consists of copolymerizing the sub
strate (gelatin, casein, collagen, etc.) on the same gel for use in
213
214 Jéssyka Fernanda Santiago Monte et al.
electrophoresis. The sample buffer is designed to increase sample

viscosity, provide a tracking dye, denaturing molecules, and con
trol the pH of the sample. Thereafter, the sample is fractionated by
electrophoresis in an electric field. Metalloproteinases present in
the sample are denatured by SDS, but are not reduced. After the
run, the proteases separated within the gel are renatured by
repeated washing with a nonionic detergent such as Triton X-100,
which replaces the SDS on the gel. The gel is then incubated in a
suitable buffer, allowing the renatured protease perform digestion
of the substrate in the surrounding region of its electrophoresis
position. These areas are visualized by staining the gel with
Coomassie blue, where the gel turns blue and digestion areas
appear as clearer zones. Metalloproteinases are then identified by
comparing the digested areas with molecular weight standards
and/or by inhibition tests with specific inhibitors of certain classes
of metalloproteinases [4].
Thus, zymography is by far the most common test used for the
detection of metalloproteinases activity; besides being very effective,
it has several advantages compared to other techniques of biologi
cal molecules, namely: (a) it does not require antibody, making it
relatively inexpensive, (b) the protein separation by molecular mass
and nonreducing electrophoretic migration confirms visually the
identity of the enzyme, (c) densitometry may be used for quantita
tive analysis, (d) there is the ability to distinguish the presence of
latent and active enzymes isoforms, (e) its relative ease of perfor
mance, (f) the development of different specific assay using differ
ent protein substrates for enzymatic cleavage, and (g) it provides a
much more sensitive test than immunoblotting for quantifying
these metalloproteinases [3, 5].
Using the zymography technique, it is possible to characterize
metalloproteinases in a wide variety of cells, tissues, and parasites
(Figs. 1 and 2). Given that a better understanding of the interac
tions established between parasites and their respective vertebrate
host is needed, the biochemical characterization of metalloprotein
ases may be a way to achieve this. A set of enzymes is known to be
common to Trypanosomatids, such as Leishmania spp. (etiological
agent of Leishmaniasis in human and animal), Trypanosoma cruzi
(etiological agent of Chagas diseases or American trypanosomia
sis), and Trypanosoma brucei (responsible for the sleeping sickness
or African trypanosomiasis). All these are involved in several aspects
of pathogenesis of neglected tropical diseases.
In the context of infections caused by trypanosomatids, we can
use the zymography assay for targeting the following issues: (a)
What are the different zymography profiles of the different species
and forms of trypanosomatids? (Fig. 1) (b) Which are the specific
substrates of metalloproteinases present in different species and
Zymography and Trypanosomiasis 215
M 1 2 3 4 5 6 7 8 9 10 11 C
kDa
190
125
80
50
40
25
Fig. 1 Zimography profile of different species of Trypanosomatids (Leishmania spp., Trypanosoma cruzi, and
Trypanosoma brucei brucei) in collagen substrate. M (marker), 1 (Leishmania amazonensis), 2 (Leishmania
guyanensis), 3 (Leishmania infantum), 4 (Leishmania shawi), 5 (epimastigote Trypanosoma cruzi strain Y), 6
(epimastigote Trypanosoma cruzi strain Bol), 7 (epimastigote Trypanosoma cruzi strain QMM5), 8 (trypomasti-
gote Trypanosoma cruzi strain Y), 9 (trypomastigote Trypanososoma cruzi Bol Strain), 10 (trypomastigote
Trypanosoma cruzi strain QMM5), 11 (bloodstreams Trypanosoma brucei brucei), and positive control (purified
collagenase). Image obtained by Monteiro, 2015 [6]
kDa kDa
t0 t5 t8 t13 t20 t0 t5 t8 t13 t20
80 80
50 50
20 20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 A B C D E F G H I J K L M N O
Fig. 2 Zimography profile (SDS-PAGE gelatin substrate) of spleens and livers samples obtained from infected
mice by Trypanosoma brucei brucei parasites. Image obtained by Gonçalves, 2011 [7]. Days postinfection
(from T0 to T20). Lanes from 1 to 15 (spleen samples) and from A to O (liver samples)
forms of trypanosomatids? (c) During the interaction of parasites

with different cell types and tissues, which are the endogenous
metalloproteinases produced as a consequence of host–parasite
interaction? (Fig. 2) (d) Which types of molecules can be used to
inhibit enzyme activity by zymography (i.e., screening for novel
metalloproteinase inhibitors)?
Indeed, numerous other scientific questions can be answered
by zymography regarding the activation process, regulation, and
interaction of parasites metalloproteinase involved in the biology
and pathogenesis of the parasite–host interaction (Fig. 2).
2 Materials
2.1 Zymography 1. Have prepared your electrophoresis system (casting mount,

glass plates with 1.5 mm spacers, combs, power supply, and
gel spatula); blotting paper; ice. Gel loading Pipette Tips and
Pipettes; 15- and 50-mL polypropylene centrifuge tubes, sterile;
Plastic gel wash containers. Agitator (lab shaker, rocking
platform). Humidified incubator set to 37 °C.
2. Prepare all solutions using deionized water and analytical
grade reagents. Prepare and store all reagents at room tem
perature (unless indicated otherwise).
3. Running Gel: Deionized water, substrate (gelatin, casein,
collagen, etc.), 30% acrylamide-bis solution (30% acrylamide
+ 0.8% bis-acrylamide), 1.5 M Tris–HCl pH 8.8, 10% sodium
dodecyl sulphate (SDS, w/v), 30% ammonium persulphate
(APS) (w/v) and N,N,N′,N′-tetramethyl-ethylenediamine-
TEMED (see Note 1).
4. Stacking Gel: Deionized water, bis-acrylamide (30% acryl
amide + 0.8% Bis-acrylamide), 0.5 M Tris–HCl pH 6.8, 10%
SDS (w/v), 30% APS (w/v), and TEMED.
5. Running buffer: 25 mM Tris, 192 mM glycine, and 0.1% SDS
(w/v) (see Note 2).
6. Sample buffer: 62.5 mM Tris–HCl, pH 6.8; 5% glycerol (v/v);
4% (p/v) SDS; 0.01% bromophenol blue (w/v).
7. Renaturing solution: 2.5% Triton X-100 (v/v).
8. Development solution: 50 mM Tris–HCl pH 7.5, 200 mM
NaCl, 5 mM CaCl2, and 0.02% Brij-35 (v/v).
9. Staining solution: Coomassie Brilliant Blue (1 g of Coomassie
Brilliant Blue; 40% ethanol and 10% acetic acid glacial per 1 L).
10. Destaining solution: 10% methanol and 5% acetic acid glacial.
3 Methods
3.1 Preparation 1. Each gel results from the juxtaposition of a stacking gel (without
of Gels substrate) to running gel (with substrate). Thus, the preparation
should be a 10% acrylamide gel with copolymerized substrate
followed by a 4% acrylamide gel without substrate, stacking gel.
Table 1 describes a resume of composition of each gel.
3.2 Ten Percent 1. Weigh 0.05 g of gelatin and dissolve completely in 5 mL of
Acrylamide Gel deionized water previously heated.
with Gelatin Substrate 2. Add to a centrifuge tube, 4.1 mL of the solution prepared in
at 4.1% (w/v) previous point, 3.3 mL of bis-acrylamide, 2.5 mL of 1.5 M
Table 1
Amounts for the preparation of running and stacking gels
Running gel Stacking gel
10% acrylamide 4% acrylamide

4.1 mL Deionized water + substrate Deionized water 2.5 mL
3.3 mL 30% acrylamide + 0.8% Bis-acrylamide 450 μL
2.5 mL 1.5 M Tris–HCl pH 8.8 0.5 M Tris–HCl pH 6.8 333 μL
100 μL 10% SDS 33 μL
50 μL 30% APS 16.6 μL
5 μL TEMED 4.7 μL
Tris–HCl pH 8.8, 100 μL of 10% SDS, 50 μL of APS 30%, and

5 μL TEMED (see Note 3).
3. Transfer rapidly 8 mL of the above solution to the glass plates
where it will be polymerize. Cover the surface of the gel with
distilled water to eliminate air bubbles and isolate it from
oxygen (in order to accelerate polymerization).
4. Remove the remaining water after polymerization and add the
stacking gel.
3.3 Ten Percent 1. Using a 15 mL centrifuge tube, dilute 1 mL of collagen 0.04%
Acrylamide Gel (v/v) collagen (Collagen, type I solution from rat tail—com
with Collagen mercially available) in 3.1 mL of deionized water.
Substrate at 10% 2. Add, to the above solution, 3.3 mL of bis-acrylamide, 2.5 mL
of 1.5 M Tris–HCl pH 8.8, 100 μL of 10% SDS, 50 μL of 30%
of APS, and 5 μL TEMED (see Note 3).
3. Transfer rapidly 8 mL of the above solution to the glass plates,
where it will be polymerized.
4. Cover the gel surface with deionized water to eliminate air
bubbles.
5. Remove the remaining water and add the stacking gel.
3.4 Acrylamide 4% 1. Mix 2.5 mL deionized water, 450 μL bis-acrylamide, 333 μL

Without Substrate of 0.5 M Tris–HCl pH 6.8, 33 μL of 10% SDS, 16.6 μL 30 μL
(Stacking Gel) 4.7% APS, and TEMED (see Note 3).
2. Add quickly this solution to the running gel already polymerized
in glass plates.
3. Fit into the glass holder the plastic comb that will shape the
wells (you may choose among single-well, 10-well, or 15-well
comb) plunging into the stacking gel still in its liquid state.
3.5 Preparation 1. Samples must be thawed on ice. Prior to running the gel,
of Samples adjust the concentration of each sample (1:1) in sample buffer
for zymography.
3.6 Separation 1. Proceed to fit the glass plates in the electrode support (see
of Proteins Note 4).
by Electrophoresis 2. Complete assembly of the electrophoresis system.
3. Add running buffer at 4 °C, between the glasses. Fill the cham
ber, to the top surrounding the gel according to manufacturer
specifications.
4. Remove carefully combs of the gels. Remove any air bubble
present. Add samples into each well.
5. Complete the assembly of the electrophoresis system. Cover
the tank with the lid. Connect to power source.
6. Run at 80 V and 4 °C (see Note 5).
7. After sample migration to the running gel, the voltage can be
raised to 100 V (see Note 6).
3.7 Renaturing 1. After electrophoresis, gels are carefully removed from their
and Developing glass plates using a gel knife (scalpel or a spatula) and place
the Gel into a container with 50 mL renaturing solution. Cut a corner
to mark the gel’s direction and incubate during 1 h at 37 °C.
2. Remove the renaturing solution and add 50 mL development
solution to the gel. Incubate overnight at 37 °C.
3. Remove the development solution and rinse gel briefly with
deionized water. Proceed to staining with 50 mL Coomassie
Brilliant Blue, for approximately 20 min under gentle agitation
at room temperature.
4. Finally, the gels must be transferred into a destaining solution.
At this stage, the colorant must remain on the entire surface of
the gel except in areas where their enzymatic activity is capable
of degrading the substrate incorporated into the gel. Thus,
enzymes capable of decomposing the substrate should stand as
white bands on a blue background. Figures 1 and 2 show the
different zymogram profiles with collagen or with gelatine,
using extract from different species of Trypanosomatids
(Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei
brucei).
All extracts were enzymatically active in colagen substrates and

each parasite evidenced a specific zymographic profile. An enzyme
with a molecular mass of 50–80 kDa, possibly the 63 kDa glycopro
tein, was detected in Leishmania species. Bands of approximately 40
and 20 kDa with enzymatic activity were observed in extracts of
T. cruzi and T. B. brucei, respectively. Given the biological importance

of metalloproteases, clarifying their role in the establishment of infec
tion and in the survival of tripanosomatids may constitute an oppor
tunity to identify new targets and, consequently, for the design and
development of new therapeutic and prophylactic strategies.
4 Notes
1. Thirty percent acrylamide-bis solution is stored in a bottle

wrapped with aluminum foil at 4 °C. TEMED and Tris–HCl
pH 8.8 and pH 6.8 is stored at 4 °C. Ammonium persulfate,
APS: 30% solution in water in large batches (10 mL) frozen
in aliquots (for greater day-to-day reproducibility) and used
indefinitely.
2. Simple method of preparing running buffer: Prepare 10×
native buffer (0.25 M Tris, 1.92 M glycine, and 0.1% SDS)
and make it to 1 L with water. Dilute 100 mL of 10× native
buffer to 900 mL with water. Based on the manufacturer’s
recommendations, the pH of the running buffer must not be
adjusted. Its correct preparation is, per se, enough to achieve
the desired pH.
3. The higher the percentage of PSA and TEMED, the higher
will be the speed of polymerization. Their concentrations
should not change too much, so as not to alter the concentra
tions of the remaining components of the gel.
4. The separation of proteins in gels containing different substrates
should be held in different electrophoresis runs.
5. If the dye front is yellow rather than blue, proceed to check the
pH of all solutions, especially the running buffer solution.
6. Monitor the progress of the migration by using the bromophe
nol blue included in the loading buffer as an indicator. Let the
gel run until the indicator dye reaches the bottom of the gels.
Acknowledgments
This work was supported by Capes—Brazil (Bolsa Jovem Talento—

Grant 019/2013) and by Global Health and Tropical Medicine
(GHTM-UID/multi/04413/2013). We wish to thank Dr. João
Aristeu da Rosa from Faculdade de Ciências Farmacêuticas de
Araraquara—Brazil, who isolated the QMM5 strain of Trypanosoma
cruzi parasite.
References
1. Kleiner DE, Stetler-Stevenson WG (1994) Trypanosoma brucei brucei parasites. Protein
Quantitative zymography: detection of pico J 29:283–289
gram quantities of gelatinases. Anal Biochem 5. Wilder CL, Park KY, Keegan PM, Platt MO
218:325–329 (2011) Manipulating substrate and pH in
2. Heussen C, Dowdle EB (1980) Electrophoretic zymography protocols selectively distinguishes
analysis of plasminogen activators in polyacryl cathepsins K, L, S, and V activity in cells and tis
amide gels containing sodium dodecyl sulfate and sues. Arch Biochem Biophys 516:52–57
copolymerized substrates. Anal Biochem 102:196 6. Monteiro JPMFL (2015) Caracterização bio
3. Frankowski H, YH G, Heo JH, Milner R, del química, molecular e propriedades biológicas de
Zoppo G (2012) Use of gel zymography to diferentes metaloproteinases de Tripansomatídeos
examine matrix metalloproteinase (gelatinase) (Trypanosoma brucei e Leishmania spp.).
expression in brain tissue or in primary glial cul Dissertation, New University of Lisbon-IHMT
tures. Methods Mol Biol 814:221–233 7. Gonçalves D (2011) Efeito da minociclina em
4. De Sousa KP, Atouguia J, Silva MS (2010) Mus musculus infectados com Trypanosoma bru-
Partial biochemical characterization of a metal cei brucei. Dissertation, New University of
loproteinase from the bloodstream forms of Lisbon-IHMT
Chapter 21
Zymography in Multiwells for Quality Assessment

of Proteinases
Ambili Mechoor and Madathiparambil G. Madanan
Abstract
Zymography is a well-standardized protocol for the qualitative assessment and analysis of proteinases
under specified conditions. However, analysis of a large number of samples simultaneously becomes a
challenge when the zymography is carried out by the usual protocol of electrophoresis. This can be over-
come by assaying the matrix-degrading proteinases in substrate-impregnated gels in multiwells. Enzymes
are copolymerized with 300 mL of 10% acrylamide impregnated with gelatin substrate and incubated for 16 h.
The gels are then stained with Coomassie blue, destained with water, and visualized with the naked eye.
The intensity; if needed can be measured with a densitometer or gel documentation system. This method
has been tested for bacterial collagenases as well as some matrix-degrading metalloproteinases that were
purified from rat mammary gland. It can also be used to characterize the enzymes with respect to the type
and concentration of the cations required for activity and the role of other regulatory molecules that may
affect the enzyme activity. The added advantage of this method is that the electrophoresis set up and elec-
tricity is not needed for the procedure.
Key words Extracellular matrix, Proteinases, Collagenase, Multiwells, Zymography
1 Introduction
The extracellular matrix (ECM) plays an important role in regulating

tissue-specific function apart from providing structural support
and tensile strength to the tissue. This is brought about by provid-
ing substrates and pathways for cell adhesion, cell migration, and
by regulating differentiated cellular function and metabolic activity
directly or indirectly [1, 2]. Remodeling of the ECM occurs in
adult tissues during mammary gland involution, hepatic regenera-
tion, uterine involution, etc. The diverse forms of ECM include
the tough elastic framework of tendons, cartilage and bone, the
crystal like cornea of the eye, and supportive elements of epithelia
as well. The various macromolecular components of the ECM include
a wide variety of collagens, elastins, glycoproteins, proteoglycans, and
complex carbohydrates. Different types of ECM are comprised of
different sets of macromolecules [3–5]. ECM degradation and tissue
221
222 Ambili Mechoor and Madathiparambil G. Madanan
remodelling take place both in normal physiological conditions

and in pathological states. One of the major players in degradation
and remodelling of the ECM are a group of zinc-dependent pro-
teinases which are generally known as Matrix metalloproteinases
(MMPs) [6].
The interaction of cells with their ECM is crucial in maintaining
a differentiated phenotype. These interactions are altered during
tissue regeneration, repair, and in pathological conditions. These
interactions are governed by a group of enzymes called MMPs.
They are zinc-containing endopeptidases that degrade extracellular
matrix proteins during tissue morphogenesis and remodeling in
mammary gland involution [7], liver regeneration, wound healing
and are associated with tumor angiogenesis, invasion and metasta-
sis, arthritis, and atherosclerosis [8–11]. MMP expression is regu-
lated at the transcriptional level by growth factors, hormones [12],
cytokines, and cell–cell matrix interactions [13]. Extracellular
MMPs are activated by proteolytic cleavage of their NH2 terminal
domains and are inhibited by noncovalent 1:1 stochiometric inter-
action with tissue inhibitors of metalloproteinases (TIMPs) [14].
Many of these MMPs are not expressed constitutively in vivo, but
rather are induced in response to cytokines, growth factors, hor-
mones, oncogenes, etc. Apart from these, they are regulated at the
transcriptional level as well as depending on the type of various tis-
sues involved. Most of these MMPs are produced as proenzyme
which needs activation to act on the specific substrate. Besides,
TIMPs also play a key role in MMP regulation. Classification of
MMPs is based on their specificity of substrate and is classified
into collagenases, gelatinases, stromelysins, and matrilysins and
membrane-type MMPs [15]. They show affinity to different metal
ions based on which their activity can be inhibited.
Many pathogenic organisms synthesize and secrete a wide
array of proteinases, of which the most common are metallopro-
teinases, particularly membrane-bound forms, which possess addi-
tional functional domains compared with secreted forms [16]. In
pathogens, also metalloproteinases play key roles in degradation of
ECM components during their invasion and pathogenesis. Some
pathogens activate host proteinase to degrade the host ECM at
their convenience [17, 18]. In such cases, these enzymes work
against host proteinase cascades, cytokine networks, extracellular
matrix components, and host enzyme inhibitor interactions [19].
But many pathogens produce their own proteinases [20]. Bacterial
proteases may be secretory forms or membrane-bound forms [21].
These proteases are used for the degradation of ECM in order to
facilitate their smooth invasion and makes pathogenesis [22]. They
are considered to be having role in invasion of the pathogen and
pathogenesis.
The conventional zymography method involves electropho-
retic separation of proteins in a substrate-impregnated acrylamide
Simple Zymographic Detection of Proteinases 223
Fig. 1 Increasing concentration of bacterial collagenase in a total volume of 100 μL (200 mM phosphate buffer
pH 7.5) was copolymerized with 300 μL of acrylamide in the wells of a 16 mm multiwell plate. The reaction
was allowed to take place for about 1 h. The gels were released and treated with substrate buffer and
analyzed as described in the procedure. Control without enzyme (1), 0.02 units (2), 0.2 unit (3), and 20 units
(4) of collagenase was used
gel and the incubation in buffers which is necessary for the activity
of these matrix-degrading enzymes. As many of these proteinase
activities are specific to their ECM substrates and activators,
requirement of metal ions for activity and pH conditions plays a
significant role. Their characterization is also based on scaling their
activity under such conditions. The analysis of a large number of
samples simultaneously under different conditions becomes a chal-
lenge when the zymography is carried out by the usual protocol of
electrophoresis. This is mainly due to the cumbersome procedure
and limited number of samples which can be loaded on the gel.
This can be overcome by assaying the matrix-degrading protein-
ases in substrate-impregnated gels in multiwells (Fig. 1). The effect
of different ions and its concentration dependence can also be
studied using this method by keeping the enzyme and substrate
concentration at the optimal levels. This method can be further
extended to convenient study of the effect of various regulatory
molecules that affect the enzyme activity and their concentration
dependence on the enzyme activity (Fig. 2) [23].
2 Materials
All solutions should be prepared using deionized water at 25 °C

and analytical grade reagents. The following materials are needed:
Gelatin from bovine skin, Triton X-100, Chondroitin sulphate A,
Chondroitin sulphate C, Heparin, Hyaluronic acid, Sodium azide.
The matrix-degrading enzymes, viz., Gelatinases, were purified
from involuting mouse mammary gland in the laboratory. Besides,
16 mm multiwell plates, incubator (for incubating the gel trays for
18 h at 37 °C), and a shaker (rocking or rotary shaker with low
speed for staining the gel).
Fig. 2 Purified gelatinases A, B, and C were pretreated with 6 μg each of gylcosaminoglycans for 30 min at
room temperature. The treated enzymes were then copolymerized with gelatine (2 mg/mL)-impregnated poly-
acrylamide in multiwell plates. After polymerization, the gels were incubated in substrate buffer, stained with
Coomassie blue, and destained with distilled water. Untreated controls (enzyme only) (1), pretreated with
heparin (2), hyaluronic acid (3), chondrotin sulphate A (4), and chondroitin sulphate C (5). Enzyme activity
inhibition is noted in the case of samples treated with condroitin sulphate A and C
2.1 Buffers 1. Acrylamide-Bis Acrylamide Solution (30% T, 3% C): Dissolve

and Stock Solutions acrylamide 29.1 g and bis-acrylamide 0.9 g in 40 mL water.
Make up to 100 mL with water and filter through a Whatman®
Grade 1 filter paper. Store the stock solution at 4 °C in an
amber-colored bottle.
2. Sodium dodecyl sulfate 10%: Dissolve 10 g SDS in 80 mL of
water and make up the volume to 100 mL and store at room
temperature.
3. Ammonium persulfate (APS) 10%: Dissolve 100 mg ammo-
nium persulfate in 1 mL of water and store at 4 °C. This stock
is stable up to 2 weeks.
4. Substrate-impregnated gels: Acrylamide solution containing
the substrate is prepared by mixing 200 mg gelatin in buffer/
water, 30 mL of 30% acrylamide solution, 100 μL of TEMED,
and 1 mL of APS for 100 mL.
5. Renaturing Buffer (2.5% Triton X-100 solution): Dissolve
2.5 mL of Triton X-100 in 97.5 mL of water.
6. Stock 500 mM Tris–HCl solution: 500 mM Tris–HCl pH 7.5
is prepared by dissolving 6.06 g Tris base in 80 mL of water
and pH made up to 7.5 with HCl. Volume made up to 100 mL
with water and store at 4 °C.
7. Stock 500 mM CaCl2: Dissolve 5.54 g CaCl2 in 100 mL water

8. Substrate Buffer (50 mM Tris–HCl pH 7.5, 1% Triton X-100,
and 5 mM CaCl2). Dissolve 1 mL of Triton X-100 in 79 mL of
water. Add 10 mL of 500 mM Tris–HCl pH 7.5 and 1 mL of
500 mM CaCl2. Make it up to 100 mL. The buffer was added
with 0.02% NaN3 in order to avoid microbial contamination.
2.2 Staining 1. Coomassie Blue Stain: To make 100 mL Coomassie blue stain
mix (40 mL methanol, 10 mL acetic acid, 50 mL of water)
dissolve 10 mg Coomassie brilliant blue R-250.
2. Destaining Solution: Pure double-distilled water.
3 Methods
3.1 Sample 1. Matrix-degrading proteinases are isolated from various stages

Preparation of mammary gland involution by standard protocols of pro-
of Proteinases tein extraction [7].
and Collagenases 2. The enzymes from the mammary epithelia cells in culture are
isolated by substrate affinity using gelatin agarose beads [24].
The enzymes thus purified are used for all the experimental
protocols.
3.2 Preparation 1. The required amount of enzyme (approximately 0.02–

of Substrate- 200 units) (MMPs/Collagenases/Gelatinases) in a maximum
Impregnated Gels volume of 100 μL is pipetted out into each well of a 16 mm
in Multiwells multiwell plate (see Note 1).
2. Gelatin or Collagen IV (2 mg/mL) is dissolved (by heating at
70 °C) in 50 mM Tris–Cl pH 7.5. Acrylamide 10% was made up
to 40 mL with Tris–HCl buffer and mixed well. 300 μL of this
mixture is pipetted out quickly into each well as a layer over the
evenly spread enzyme (see Note 2). This leads to a final acryl-
amide concentration of 7.5%. The gels are allowed to polymerize
for about 1 h. The gels are then carefully detached from the
plastic and incubated with 1 mL of Tris–HCl buffer [50 mM
Tris–HCl buffer pH 7.5, 5 mM CaCl2, and 0.02% NaN3] for
16 h at 37 °C.
3. The buffer is then carefully removed and the gels are stained
with Coomassie brilliant blue for 15 min and destained with
water for 2 h. The substrate degradation can be visualized with
the naked eye and, if needed, can be quantitated using a
densitometer.
3.3 Preparation 1. Add different amounts (approximately 0.02–200 units)

of Multiwell Gel (concentration dependence as in legend from Fig. 1) of enzyme
(MMPs/Collagenases/Gelatinases) dissolved in a final volume
of 100 μL of 50 mM Tris–HCl pH 7.5 per well. We have used

different amounts of collagenase dissolved in the same buffer.
2. Add 300 μL substrate-containing acrylamide solution carefully
over the evenly distributed enzyme in the well. This leads to a
final acrylamide concentration of 7.5%.
3. The gels are then allowed to polymerize for about 1 h.
4. After polymerization, the gels are carefully detached from the
plastic and incubated with 1 mL of substrate buffer for 16 h at
37 °C (see Notes 1–3).
3.4 Pretreatment 1. In order to study the effect of various parameters, viz., ions, other
of Enzymes ECM components, activators, inhibitors, etc., the enzyme
and Quality solution is incubated with the component for about 30 min at
Assessment room temperature (25 °C) before subjecting it to polymerization
with substrate-impregnated acrylamide solution (see Note 4).
2. The incubation buffer can be modified by adding different
activators and inhibitors of choice to study their effect.
3. As shown in Fig. 2, we have assessed an untreated sample as nega-
tive control (enzyme only) (1), enzyme pretreated with heparin
(2), hyaluronic acid (3), chondrotin sulphate A (4), and chon-
droitin sulphate C (5). Enzyme activity inhibition is noted in the
case of samples treated with chondroitin sulphate A and C.
3.5 Quantification 1. Densitometry or gel documentation scanning can be used to

Using Densitometry or scale the activity between different samples.
Gel Documentation 2. The gels were kept in the scanner and the peak density across
Scanning the diameter of round gels will be taken as measure of activity
(for more accuracy, an average of diameters at multiple axis
may be used) (see Note 5).
3. An interpolation against known peak density of standard
samples with known activity or concentration may be used to
measure the unknown sample (see Note 6).
4 Notes
1. The enzyme assay has to be done by other standard protocols,

the optimal concentrations calculated, and has to be brought
to the necessary concentration according to the experiment.
Care has to be taken that the required concentration does not
exceed a volume of 100 μL. One enzyme unit is defined as the
amount that catalyzes the conversion of 1 μmol of substrate
(gelatin) per min at 25 °C.
2. The multiwell plate has to be placed on an even surface and
utmost care has to be taken for the even uniform spreading of
the enzyme solution in each of the wells. Appropriate controls
are taken in the same volume. For instance, a standard proteinase

whose activity has been established can act as a positive control.
In our case, the standard taken was bacterial gelatinase.
3. Sodium azide in low concentrations ~0.01–0.02% is used as a
preservative to prevent fungal and bacterial growth in buffers.
Excess concentration can be toxic to the system and can pos-
sibly inactivate the enzyme.
4. The cations used in the study were 2 mM each of Ca2+, Zn2+,
Mg2+, and EDTA. The inhibitors/modulators used in this
study were 1 mM each of FDNB (1-fluoro-2,4-dinitroben-
zene) and iodoacetate. These molecules are known to inhibit
MMP activity. In case of other ECM components, we had
used heparin, hyaluronic acid, chondroitin sulfate A, and
chondroitin sulfate C (1–6 μg each).
5. The gels are removed from the multiwell plate and scanned
individually. A slight pressure with the end of the spatula
releases the gels from the plate.
6. Among other applications and further advantages of multiwell
zymography: similarly other purified proteinases from eukary-
otes and bacteria may be used [25].
Acknowledgments
The author A.M. is grateful to Indian Council of Medical Research

for receiving ICMR-JRF during the conduction of the study.
References
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2. Hay ED (1993) Extracellular matrix alters epi- In: Hooper NM (ed) Zinc metalloproteases in
thelial differentiation. Curr Opin Cell Biol health and disease. Taylor and Francis, London,
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3. Yamada KM (1983) Cell surface interactions 9. Sternlicht MD, Lochter A, Sympson CJ, Huey
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tor with erythroid potentiating activity: evi- (2011) A zymography analysis of proteinase
dence for inducible and sonstiturive activity present in Leptospira. Curr Microbiol
transcripts. Nucleic Acids Res 14: 62:917–922
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their inhibitors in matrix remodeling. Trends (2011) Presence of 46 kDa gelatinase on the
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Chapter 22
Visualization of Enzyme Activities in Earthworm Biopores

by In Situ Soil Zymography
Bahar S. Razavi, Duyen Hoang, and Yakov Kuzyakov
Abstract
Earthworms produce biopores with strongly increased microbial and enzyme activities and consequently
they form microbial hotspots in soil. In extremely dynamic microhabitats and hotspots such as earthworm
biopores, the in situ enzyme activities are a footprint of process rates and complex biotic interactions. The
effect of earthworms on enzyme activities inside biopores, relative to earthworm-free soil, can be visualized
by in situ soil zymography. Here, we describe the details of the approach and discuss its advantages and
limitations. Direct zymography provides high spatial resolution for quantitative images of enzyme activities
in biopores.
Key words Earthworm, Drilosphere, Spatial distribution, Enzymes activity, Microbial hotspots,
Zymography
1 Introduction
Extracellular enzymes are involved in innumerable biogeochemical

processes and are central for the processing, stabilization, and desta-
bilization of soil organic matter and nutrient cycling in terrestrial
ecosystems [1, 2]. Extracellular enzyme activities are most strongly
controlled by the concentration of enzymes, their confirmation, and
corresponding substrates [3]. The abundance of C-, N-, and
P-degrading enzymes in soils is controlled by numerous factors
including microbial biomass, community composition, substrate
availability, microclimate, and stoichiometric demands [4]. Most
enzymes are assumed to originate from microorganisms; however,
plant roots and soil animals can contribute strongly to enzyme
abundance either directly, by enzyme production, or indirectly, by
releasing organic substrates that stimulate microorganisms [5].
Earthworms, which are the most important soil dwelling ani-
mals, play an important role by mixing soil materials, aggregating
soil particles, and digesting plant litter [6, 7]. The network of bio-
pores formed by earthworms is termed drilosphere and is among
229
230 Bahar S. Razavi et al.
the most important microbial hotspots in soil [8]. The high microbial
activity in the drilosphere is explained by the input of labile organic
materials and their mechanical and microbial processing within
earthworm gut in the well-aerated and stable biopore structure.
High microbial activities, in turn, accelerate the transformation
and mineralization of carbon (C) and nutrients, such as nitrogen (N)
and phosphorus (P).
Determination of enzyme activities by fluorogenically labeled
substrates is frequently applied in soil and rhizosphere studies.
Visualization of enzyme activity inside burrow linings in undis-
turbed samples would strongly improve our understanding of
earthworms’ effects on enzyme activities [8, 9]. The zymography
technique [10] has been applied in various scientific fields, such as
medicine, physiology, and biochemistry. With zymography, the
conversion of the substrate to a reaction product can be visualized
nondestructively [11]. It yields spatially resolved quantitative and
qualitative information about hydrolase activities on a surface [11].
Zymography has previously been adapted to visualize the spatial
and temporal dynamics of enzyme activities in soil with living and
dead roots [12–14]. However, visualization of enzyme activities
inside earthworms’ habitats was remained a challenge. Recently,
this technique has been developed to be applied for quantitative
imaging of enzyme activities in biopores [15].
The objectives of this chapter are to describe zymography
application to visualize enzyme distribution inside earthworm bio-
pores for nondestructive and quantitative assessment of enzyme
activities both under laboratory and field conditions.
2 Materials
1. Dissolved each of substrates separately in universal buffer to a

concentration of 12 mM. [This technique can be used for
various hydrolytic enzymes (Table 1) (see Notes 1–4).]
2. Buffer preparation:
(a) 4-Morpholineethane sulphonic acid (MES), (0.1 M), for
4-methylumbelliferone (MUF) substrate: Weigh 20.673 g
MES (206.73 g/mol), put in a 1000 mL volumetric flask
and add distilled water. Desired pH would be 6.5 [16].
(b) TRIZMA (0.05 M) buffer for 4-methylcoumarin (AMC)
substrate: Weigh 0.985 g Tris-Base (α-α-α-Tris-
(hydroxymethyl)-methylamin) and 2.66 g Tris–HCl (Tris
(hydroxymethyl) aminomethane-hydrochloride buffer,
0.036 M) and put them in one 500 mL volumetric flask
and add distilled water, to reach 500 mL. Desired pH
would be 7.2 [17] (see Note 5).
In Situ Soil Zymography 231
Table 1
Summary of selected enzymes, their main ecological functions, and substrate proxies
Enzyme Synthetic substratea Enzyme function

β-Glucosidase MUF-β-d-glucopyranoside Releases glucose from cellulose
Cellobiohydrolase MUF-β-d-cellobioside Releases disaccharides from cellulose
Xylanase MUF-β-d-xylopyranoside Releases xylose from hemicellulose
Chitinase MUF-N-acetyl-β-d-glucosaminide Releases N-acetyl glucosamine from
chitin
Phosphatase MUF-phosphate Releases attached phosphate groups
Leucine-aminopeptidase l-Leucine-7-amido-AMC Hydrolysis of the peptide bonds
Tyrosine-aminopeptidase l-Tyrosine-7-amido-AMC Hydrolysis of the peptide bonds
For each synthetic substrate, the fluorescent dye 4-methylumbelliferone (MUF) and 4-methylcoumarin (AMC) are
a
presented
3. Polyamide membrane (0.45 μm pore size) should be cut into

pieces of biopore sizes and shapes (e.g., 5 × 10 mm) [15].
Thereafter, membrane should be soaked in prepared substrate
solutions (see Note 3).
2.1 Experiment 1. Use a transparent (or opaque) plastic box (size variable
Under Lab Condition depending on the objectives) for the experiment; with a
(Rhizobox Preparation) removable front panel that could be easily opened without
affecting the earthworm habitats (see Notes 6 and 7).
2. Before filling the boxes with soil, lay a sand layer on the bot-
tom of the pots for drainage, to prevent water saturation
(see Note 8).
3. Regulate water content and keep it stable at 30% of soil dry
weight during whole experiment.
4. Depending on the objectives, place number of mature earth-
worms (three or more), by different length (5–10 cm long) in
each pot.
5. Prepare similar extra box without earthworm as a control box.
Control box will comfort your further comparison between
biopores and bulk soil.
6. If the effect of plant roots is also going to be considered in the
study, plant seedling in the soil simultaneously with
earthworms.
7. Keep the boxes under a stabled temperature of 18 ± 1 °C and a
daily light cycle of 16 h, with light intensity of 300 μmol/m2/s.
8. After 2 weeks that earthworms formed biopores, samples are
ready for zymography (Fig. 1).
Fig. 1 An example of the rhizobox and biopores formed by earthworms during

2 weeks
2.2 Experiment 1. The biopore-window consists of a transparent acrylic sheet

Under Field Condition: (3–5 mm thick).
Biopore-Window 2. The window can be 30 × 30 cm2 or 20 × 20 cm2.
System (Rhizotrons
3. It is recommended to install rhizotrons in field simultaneously
Installation) with plant sowing (if effect of plant roots would be considered
in the study).
4. Install rhizotrons at an angle of 90° to the soil surface, and fix
it in place by two vertical steel rods and backfill it with soil to
remove air gaps.
5. Place some earthworms (2, 3, to) behind the rhizotron to
increase the number of burrows.
6. Backfill rhizotron with the soil (Fig. 2).
7. After 2–3 weeks, samples are ready for zymography (Fig. 3).
3 Methods
3.1 Experiment 1. Open rhizobox from one side.

Under Lab Condition 2. The size of membranes should be adjusted to the rhizobox.
3. Additional membranes should be cut to pieces of the biopore
size.
Fig. 2 Scheme of biopore-window under field conditions. Rhizotrone should be

fixed, while the dug-out soil will be placed back
Fig. 3 An example of the biopores formed by earthworms under field condition

within 4 weeks after biopore-window establishment
4. Substrate-soaked small pieces of membrane will be separately

placed inside biopores.
5. Then they should be filled with flint glass beads (0.5 mm).
6. Soft plastic stuffing should be inserted between the glass beads
and membrane to ensure the proper membrane attachment to
the biopore wall.
Fig. 4 The polyamide membrane is sandwiched between soil surface and glass plate (under lab condition) or
rhizotrone (under field condition) and incubated for 1 h
7. At the same time, a large membrane (matching the box side)

will be attached to the whole exposed soil surface.
8. The saturated membranes should be incubated at the soil
surface for 1 h.
9. Cover membrane with a glass plate during incubation (to prevent
evaporation) (Fig. 4).
10. After incubation, few soil particles attached to membranes
should be gently removed using tweezers.
11. The membranes will be carefully placed under UV light
with an excitation wavelength of 355 nm in a light-proofed
(dark) room.
12. Thereafter, all membranes should be photographed under UV
light.
13. If you will couple zymography with routine determination of
enzyme activity, collect biopore samples carefully from inside
earthworm burrows (see Note 9).
3.2 Experiment 1. Dig out the soil that covered the rhizotron during biopore-
Under Field Condition window establishment.
2. Carefully, remove the rhizotron without any disturbance of
biopores.
3. Cut the coated membranes into sizes adjusted for desired area.
4. Also, cut additional membranes into pieces associated with
pore size.
5. Separately, place substrate-soaked small pieces of membrane
inside biopores (Fig. 5).
6. Then fill them with flint glass beads (0.5 mm).
7. Insert soft plastic stuffing between the glass beads and mem-
brane to ensure the proper membrane attachment to the bio-
pore wall.
8. At the same time, attach a large membrane fitted for the larger
area around the biopore to the soil surface (Fig. 5).
9. Incubate the saturated membranes to the soil surface for 1 h.
10. Cover membrane with a clean rhizotron (transparent acrylic
sheet) during incubation (to prevent evaporation) (Fig. 4).
Fig. 5 An example of phosphatase activity, (a) initial zymogram of soil surface and
biopore edges; (b) zymogram of soil surface and biopore edges after image pro-
cessing; (c) zymogram of inside biopore after image processing. Side colormap is
proportional to the calibration line of MUF substrate. Red color demonstrated high
color intensity (high activity) and blue shows low color intensity (low activity).
Asterisk (*) shows another source of hotspots, i.e., root
11. After incubation, gently remove few soil particles attached to

membranes using tweezers.
12. Cover membranes from dust and to prevent evaporation and
transport them to the laboratory.
13. Place the membranes under UV light with an excitation wave-
length of 355 nm in a light-proofed (dark) room.
14. Thereafter all membranes should be photographed.
15. If you will couple zymography with routine determination of
enzyme activity, collect biopore samples carefully from inside
earthworm burrows (see Note 9).
3.3 Image Analysis For quantitative analysis of the zymogram images, calibration is
required, which relates the enzyme activities to the gray-values
3.3.1 Calibration
projected onto the zymograms.
1. Eight to ten pieces of membranes should be cut to 3 or 4 cm2
(Fig. 5).
2. Soak cut membranes in exact volume (e.g., 5 μL) of solution
of MUF or AMC with the range of concentrations. For example:
0.01, 0.2, 0.5, 1, 2, 4, 6, 10 μM (This step is necessary to
quantify your images.) (see Note 10).
3. Place the calibration membranes under UV light with an
excitation wavelength of 355 nm in a light-proofed room.
4. All the membranes will be photographed.
Fig. 6 (a) Example of roots grown in control box. (b) Initial zymogram of soil surface and roots. (c) Zymogram
of soil surface and roots after image processing (AMC-leucine aminopeptidase). Hotspots in control soil,
mainly, associated with roots
3.3.2 Image Processing Image processing and analysis could be done by any image processor
software. Here, image processing in Matlab environment would be
explained briefly [14] (see Notes 11 and 12).
1. Transform the zymograms to 16 bit grayscale images as a
matrix.
2. Make spatial referencing based on the gray-value received
from a reference object, which is fixed in all the zymograms
(see Note 13).
3. Calculate the average background gray-values through the
calibration lines at zero concentration, and subtract this
background value from all zymograms.
4. To illustrate the results, the values of the grayscale image could
be depicted in color (Figs. 5 and 6) (see Note 10).
5. Convert the pixel-wise gray-values of the zymograms to enzyme
activities using the enzyme-specific calibration function.
4 Notes
1. Sterile and autoclave all the plates, which will be used for prepa-
ration of buffer, substrate solution, and scaling function.
2. Wear gloves and use very precise scale to weight the all materials,
especially substrates.
3. It is recommended to find the proper substrate amount in
preliminary tests.
4. The substrate solution can be stored at 4 °C for 2 weeks.
However, in our laboratory we make substrate solutions fresh.
We find that it is better to prepare them fresh each time.
5. Adjust the pH, close to the soil natural condition. Above-

maintained buffer is proposed for soil with pH: 7–7.5.
6. If the earthworms are collected manually from field, place them
in a black pot with in situ soil at room temperature for 1 week,
to adapt the earthworms to the new environment.
7. Cover the boxes with aluminum foil or black paper to protect
them from the light and prevent algae growth.
8. Water content should be regulated and kept stable similar to
the original habitat.
9. Have in mind that these burrows should not be used for
zymography (because burrows cannot be considered undis-
turbed samples any more).
10. Soak cut membranes in different concentration of MUF solution
to quantify the enzyme activities of substrates that are MUF-
based, and soak cut membranes in different concentration of
AMC solution for substrates that are AMC-linked (Table 1).

11. Interpretation of obtained images: Stronger color intensity
(red color) compared to control soil illustrates the hotspots
made by earthworms, which are concentrated mainly on the
edges and wall of burrow linings and also inside burrow lines
(Fig. 5). In comparison, hotspots (red color) in control soil are
mainly associated with the root (rhizosphere) (Fig. 6).
12. Calculation of active sites (hotspots area): This step can be done
after converting the pixel-wise gray-values of the zymograms to
enzyme activities by using the enzyme-specific calibration func-
tion. Thereafter, segment the hotspot length and visible area
based on different categories of activity (no activity, low, medium,
high, and very high), [15]. To confirm the boundaries, you can
apply one way analysis of variance (ANOVA) to assess the signifi-
cant differences between independent variables (adjacent pixels).
Calculate and compare these segments as the percentage of total
hotspot area (percentage of hotspot area per cm2) to the entire
image (total surface of boxes, inside biopores, and control soil).
13. Blank sides of the sample could be considered as the referenc-
ing point (as background). Otherwise on each image, 2 cm2
black color sheet could be placed, so that you will have same
color scale for all zymograms.
Acknowledgments
We gratefully acknowledge the German Academic Exchange

Service (DAAD) for supporting B.S.R. and Vietnamese govern-
ment for supporting D.H. Authors thank Ali Feizi for linguistic
help. The study was supported by the German Science Foundation
(DFG) within project KU 1184/29-1.
References
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Chapter 23
Multiplex Cathepsin Zymography to Detect Amounts

of Active Cathepsins K, L, S, and V
Manu O. Platt
Abstract
Cysteine cathepsins are powerful proteases that can degrade other proteins, among which are the extracel-
lular matrix proteins collagen and elastin. Multiplex cathepsin zymography is an assay that can quantify the
amount of active cathepsins in a cell or tissue preparation. This method works for measuring the amounts
of active cathepsins K, L, S, and V in a cell or tissue preparation without requiring the use of antibodies for
specific identification which tremendously reduces cost. This chapter will demonstrate the utility and inter-
pretation of this method with mammalian cells and tissue to quantify amounts of active cathepsins K, L, S,
and V without complicating signals of the procathepsin. Multiplex cathepsin zymography has many
advantages: (1) it separates cathepsins K, L, S, and V by electrophoretic migration distance, (2) allows
visual confirmation of cathepsin identity, (3) does not detect procathepsins, and (4) can be quantified with
densitometry.
Key words Cathepsins, Proteases, Cancer, Cardiovascular disease, Enzymology
1 Introduction
Cathepsins K, L, S, and V are members of the lysosomal cysteine

cathepsin family which have been implicated in a number of patho-
logical roles. Among the four, they share 60% sequence homology
[1–3], but each has unique properties and different homeostatic
functions. Cysteine cathepsins are a potent class of proteases that
have been shown to be upregulated in a number of tissue-
destructive diseases such as cancer, arthritis, osteoporosis, and ath-
erosclerosis [1, 4–9].
Multiplex cathepsin zymography is based on gelatin zymogra-
phy techniques that have been used to detect matrix metallopro-
teinases (MMPs) [10], but has been modified for specific members
of the cysteine cathepsin family [11, 12]. It has also been referred
to as substrate SDS-PAGE (sodium dodecyl sulfate polyacrylamide
gel electrophoresis) due to polymerizing a gelatin substrate in the
polyacrylamide gel meshwork. Proteins extracted from cells or
239
240 Manu O. Platt
tissue can be resolved in a nonreducing electrophoresis with a

standard Bio-Rad Mini-Protean chamber or equivalent apparatus
to separate proteins by molecular size after being partially unfolded
by SDS. Excluding beta-mercaptoethanol preserves disulfide
bridges in the cathepsin native conformation [6, 13], preventing
full linearization of the molecule, but enabling refolding after
washing out the SDS in a renaturing buffer. The gel is then equili-
brated to pH 6, which is optimal pH for their function, prior to
overnight incubation in fresh assay buffer with a freshly added
reducing agent, dithiothreitol (DTT), to provide an optimal reduc-
ing environment and pH for cathepsin activity. These refolded,
active enzymes will then cleave the gelatin substrate in the poly-
acrylamide gel at the site to which they were separated by the elec-
trophoresis such that after an overnight incubation period, the gel
can be stained with Coomassie blue, which will stain the entire gel
blue due to the cross-linked gelatin, except where the cathepsins
have degraded the gelatin presenting as cleared white bands of pro-
teolytic activity after destaining. Schematic of this entire protocol
is presented in Fig. 1. Only refolded, active cathepsins will provide
a signal that can be quantified by densitometry methods such as
NIH ImageJ. This removes the confounding signals from pro-
forms of cathepsins that would be detected by ELISA, Western
blot, and other antibody-based methods.
Multiplex cathepsin zymography has been demonstrated with
recombinant enzymes [14, 15], a number of different cell types
including endothelial cells [8, 16–19], monocytes [17], macro-
phages [20, 21], breast cancer cells [20], prostate cancer cells [22],
osteoclasts [21], and others, and also a number of homogenized
tissues including tendons, breast, lung, and cervical tumors [23],
endometriosis lesions [24], arteries [8, 18, 25–27], and heart
valves [28, 29] among others.
Fig. 1 Schematic representation of the cathepsin zymography protocol

Multiplex Cathepsin Zymography to Detect Amounts of Active Cathepsins K, L, S, and V 241
As described, this protocol will require 2 h of attention and

preparation prior to the overnight incubation step and usually
requires 10–15 μg of protein material to obtain a signal. This tech-
nique has many benefits: (1) it does not require antibodies, (2)
separation of proteins by molecular mass visually confirms cathep-
sin identity, (3) densitometry can be used for quantitative analysis,
and (4) versatility of the assay enables inhibition with small mole-
cules to corroborate the identity of the enzyme of interest.
2 Materials
2.1 SDS 1. Gelatin preparation: Dissolve 0.150 g gelatin (from pig or

Polyacrylamide Gel bovine extract) in 30 mL of warmed, deionized water to obtain
Components a final concentration of 5 mg/mL (see Note 1). Store this
gelatin solution in a 50 mL conical tube labeled with the date
2. Separating Gel Components: Add about 200 mL of deionized
water to a beaker with a magnetic stir bar and place this together
on a stir plate. Weigh 113.3 g Tris base and transfer it to the
beaker. Fill with water to 500 mL. Then adjust pH with HCl to
8.9. This solution can be stored at room temperature.
3. Stacking Gel Components: As above, prepare a beaker, stir bar,
and stir plate with 200 mL of deionized water. Weigh 19 g
Tris base and transfer it to the beaker. Fill with water to
500 mL. Then adjust pH to 6.7 with HCl. This solution can
also be stored at room temperature.
4. Acrylamide/bisacrylamide mixture at 37.5:1 ratio: Commonly
used version is ProtoGel available from National Diagnostics.
To be stored in a dark jar protected from light.
5. Sodium dodecyl sulfate (SDS): 10% solution in water (see Note 2).
6. Ammonium persulfate: 1.5% solution in water (see Note 3).
7. TEMED N, N, N′, N′-tetramethyl-ethylenediamine (TEMED):
store protected from light.
8. SDS-PAGE running buffer (10× stock): In a large 5 L beaker, fill
to about 2 L with deionized water, add a magnetic stir bar, and
place on a stir plate. Measure out 120 g Tris base, 576 g glycine,
and 40 g SDS and add to the stirring solution. Allow it to mix
until all are completely dissolved. Fill with deionized water to
4 L. Then adjust pH to 8.3 with HCl or NaOH. This can be
stored at room temperature. Dilute 100 mL of this 10× stock by
adding 900 mL deionized water in a graduated cylinder to pre-
pare a 1× working solution when ready to start electrophoresis.
9. Prestained protein ladder: Commercially available ladders with
prestained bands from 10 kDa to at least 150 kDa.
242 Manu O. Platt
2.2 Sample 1. Zymogram lysis buffer: To prepare 500 mL, dissolve the following
Preparation components into deionized water. 20 mM Tris–HCl (1.21 g);
5 mM ethylene glycol tetra-acetic acid (EGTA, 951 mg);
150 mM sodium chloride (NaCl, 4.38 g); 20 mM beta-glycerol-
phosphate (2.16 g); 10 mM sodium fluoride (NaF, 210 mg);
and sodium orthovanadate (Na3VO4, 91.95 mg). Then add
liquid detergents: 5 mL of Triton X-100 to get a final 1% v/v,
and 500 μL of Tween-20 to a final 0.1% (v/v).
2. Leupeptin: To be freshly added to the zymo lysis buffer at a
final concentration of 0.1 mM. Stock solutions of 20 mM can
be aliquoted and stored at −20 °C. Leupeptin molecular
weight is 475.59. Add 526 μL of deionized water to 5 mg of
leupeptin to make the 20 mM solution. Then, aliquot into
20 μL aliquots that can be stored at −20 °C and thawed indi-
vidually upon use. Then to prepare 1 mL of zymo lysis buffer,
add 995 μL of zymo lysis buffer and 5 μL of freshly thawed
20 mM leupeptin to be diluted 1:200 to prepare a final con-
centration of 0.1 mM.
3. 5× Sample nonreducing loading buffer: To prepare 100 mL,
weigh out and measure 0.05 g Bromophenol Blue and 10 g
SDS. Add this to 20.8 mL of 1.5 M Tris buffer, pH 6.8, and
25 mL deionized water. Then add 50 mL glycerol and mix
(see Note 4).
2.3 Renaturing To prepare 500 mL, measure out 3.94 g Tris base (65 mM) and
Buffer add to 300 mL of deionized water with a stir bar on a stir plate.
After it has dissolved, fill to 400 mL with deionized water. Then,
while still stirring, add 100 mL glycerol [20% (v/v)] to the beaker.
Then adjust pH to 7.4 and store in a labeled bottle with a screw top.
Solution can be stored at room temperature.
2.4 Assay Buffers (a) Assay buffer pH 4.0 is a sodium acetate buffer, 0.1 M with
1 mM EDTA, and freshly added 2 mM DTT. Two solutions
will need to be prepared first as stock solutions, then com-
bined at the appropriate ratios to produce a stable buffer.
Solution A: 5.755 mL glacial acetic acid, then fill to 500 mL
(0.2 M) in water.
Solution B: Measure 13.6 g of sodium acetate
(NaC2H3O2⋅3H2O) and dissolve in 300 mL of water, then add
water to fill to 500 mL to produce a 0.2 M solution (or make
appropriate weight measure calculations if using the anhy-
drous form).
For a pH 4.0 solution, mix 205 mL of Solution A with
45 mL of Solution B. Then dilute with water to a total of
500 mL. Afterward, add 2 mL of 0.25 M EDTA. This solution
will be stable. The DTT must be added fresh before incubating
with the zymogram. Test the pH to be sure that it is 4.0 either
with pH paper or a pH meter.
(b) Assay buffer pH 6.0 is a sodium phosphate buffer, 0.1 M, with

1 mM EDTA, and freshly added 2 mM DTT.
Solution C: Measure out 13.8 g NaH2PO4⋅H2O, then dis-
solve in 300 mL of water (or make appropriate weight measure
calculations if using the anhydrous form). Then fill to 500 mL
to make a 0.2 M final solution.
Solution D: Measure out 26.825 g Na2HPO4⋅7H2O, then
dissolve in 300 mL of water (or make appropriate weight mea-
sure calculations if using the anhydrous form). Then fill to
500 mL to make a 0.2 M final solution.
For a pH 6.0 solution, mix 219.25 mL of Solution C with
30.75 mL of Solution D. Then dilute with water to a total of
500 mL. Then add 2 mL of 0.25 M EDTA to make it 1 mM
EDTA. This solution will be stable. The DTT must be added
fresh before incubating with the zymogram. Test the pH to be
sure that it is 6.0 either with pH paper or a pH meter.
2.5 Staining 1. Coomassie Stain solution: To make a 1 L volume of this solu-
and Destaining tion, measure 650 mL of water, and add 100 mL acetic acid,
250 mL 2-propanol, and 0.45 g Coomassie Blue R250.
2. Destain solution: To make a 1 L volume of this solution, mea-
sure 800 mL water, and add 100 mL 2-propanol and 100 mL
acetic acid.
3 Methods

specified.
3.1 Cell Extraction 1. Add 60 μL of zymo lysis buffer to each well of a 12-well dish and
and Sample scale accordingly for larger size dishes. Goal is to get a final pro-
Preparation tein concentration greater than 1 μg/μL. If homogenizing tis-
sue, then use appropriate amount of zymo lysis buffer based on
tissue size and expected mass, still with the goal of getting final
solubilized protein concentration of greater than 1 μg/mL.
2. Perform cell extraction on ice. Use a cell scraper to ensure col-
lecting all cell extracts and collect in a microcentrifuge tube.
3. Sonicate the cell extract briefly using a probe sonicator to
ensure disruption of cell membranes.
4. Centrifuge sonicated cell extracts at high speed (>13,000 × g)
to pellet cellular debris and any insoluble fractions.
5. Remove soluble supernatant and place in a separate, new
microcentrifuge tube.
6. Perform a protein assay to determine total protein concentration
(Pierce Micro-BCA kit is recommended, but other versions
will do).
244 Manu O. Platt
7. Prepare normalized protein extract samples and add appropriate

volumes of 5× nonreducing sample buffer such that a final pro-
tein concentration of at least 1 μg/μL is obtained (see Note 5).
3.2 Substrate Gel 1. Prepare assembly and plates for gel electrophoresis, with a
Electrophoresis 0.75 mm spacer plate and a short plate. Fill with water to test
for leakage while preparing the gel solution.
2. Gather the appropriate combs to prepare wells for the 0.75 mm
thick polyacrylamide gel with cross-linked gelatin.
3. Prepare two substrate polyacrylamide standard size gels
[approximately 92 × 68 × 0.75 mm3 (width × height × thick-
ness) which are the standard Bio-Rad dimensions], mix the
following solutions in this order: 860 μL deionized water,
3.34 mL Protogel (acrylamide/bis-acrylamide), 1.6 mL 5×
separating buffer, 1.72 mL of 5 mg/mL gelatin solution,
400 μL 1.5% APS, and 80 μL 10% SDS.
4. At this point, be sure to check the plate assembly for any leaks,
and if no leaks, then pour out the water and use a tissue to
remove the remaining water in the assembly by capillary
action.
5. Then add 7.5 μL TEMED to the polyacrylamide gel solution
to initiate the polymerization reaction and quickly inject into
the plate assembly up to a level that still allows for the combs
to be inserted and for the stacking gel to be added.
6. Inject about 150 μL of butanol onto the separating gel to
make an airtight seal, remove any air bubbles in the solution,
and to create a flat line for integration with the stacking gel.
7. While the separating gel polymerizes, prepare the gel solution
for the stacking gel by mixing in this order: 1.9 mL water,
1 mL Protogel, 1 mL 5× stacking buffer, 1 mL 1.5% APS, and
50 μL 10% SDS.
8. Be sure the separating gel has polymerized by tipping the
assembly slightly to watch only the butanol layer flow and not
the polymerized gel (it will take at least 10–15 min for com-
plete polymerization).
9. At this point, be sure to remove the butanol from the gel plate
assembly first by decanting, then by adding deionized water,
and swirling it side to side to be sure that the butanol is dis-
placed above the water layer. Then decant and repeat until it
has been assured that all butanol has been removed.
10. Then prepare the comb, and add 4 μL of TEMED to the
stacking gel solution, swirl briefly, then using the pipette,
inject into the plate assembly over the separating gel. Fill the
plates and then insert the combs immediately prior to polym-
erization of the gel.
11. After polymerization of the stacking gel, carefully remove the

comb from the gels being sure not to disturb the wells, by
pulling them straight out.
12. Assemble the gels onto the electrode assembly with the short
plates on the inside (if using the Bio-Rad Mini-Tetran
apparatus).
13. Place into the electrophoresis chamber and fill with 1× SDS-
PAGE running buffer to the appropriate volume according to
the apparatus being used.
14. Carefully load equal protein amounts of samples into each lane
of the gel. Also, load a protein ladder and a positive control
(see Note 6).
15. Add a matching volume of 1× buffer to remaining wells to
ensure an even running front during the electrophoresis.
16. Attach the electrodes to the voltage box and apply 200 V for
about 1 h to separate the proteins in the sample, watching the
gel until the bromophenol blue line front reaches the bottom
of the gel (see Note 7).
3.3 Renaturing 1. After electrophoresis, carefully remove polyacrylamide gels

of Enzymes from the apparatus being sure not to rip the thin gels.
and Cleavage 2. Place them into containers with renaturing buffer, and place
of Substrate Proteins on a shaker rotating at slow to medium speed.
3. After 10 min, decant renaturing buffer, add fresh renaturing
buffer, and place back on the shaker. Repeat two more times
for a total of three times (see Note 8).
4. During this renaturation, thaw one frozen aliquot of 0.5 M
DTT in preparation of the assay buffer.
5. During the third rinse in renaturing buffer, prepare the assay
buffer (pH 6.0 for cathepsins K, L, S, and V, or pH 4.0 to
distinguish cathepsin L uniquely) by adding 200 μL of 0.5 M
DTT to 50 mL of assay buffer to get to a final concentration
of 2 mM DTT (see Note 9).
6. After the third rinse, decant the renaturing buffer and add
25 mL of the assay buffer to the container to equilibrate the
gel for 30 min (see Note 10).
7. Prepare plastic containers to incubate gels overnight with fresh
assay buffers at 37 °C. This can be done in old pipette tip
boxes with the inside racks removed, or the preferable way is
to use airtight plastic bags sealed with the plastic sealer. If
using plastic bags, then the bags can be incubated in a 37 °C
water bath overnight. If using a container, then it can be
placed inside of an incubator overnight, but agitation is rec-
ommended for best results (see Note 11).
246 Manu O. Platt
3.4 Coomassie 1. After 24 h incubation, stain with Coomassie blue for 1 h
Staining (see Note 12).
2. Rinse with deionized water.
3. Add destain buffer to observe cleared white bands on a dark
blue background indicative of cathepsin activity.
3.5 Imaging 1. Imaging of the gel can be done with a lightbox and a digital
and Densitometry camera, a scanner, or any number of types of gel imaging
apparatus such as the ImageQuant.
2. Densitometry of the signals can be performed using NIH
ImageJ, or other software that might come with your imaging
equipment, being sure to invert the image to convert the
cleared white bands on a dark band to dark bands on a white
background (see Notes 13–16 for data interpretation).
4 Notes
1. To warm the water, either use a hot plate, or for faster prepara-
tions, add 50 mL of deionized water to a small beaker and
microwave for 30 s. This will sufficiently warm the water for
quick dissolution of the gelatin. Gelatin can be used for 48 h
before it is recommended to make a fresh batch.
2. Be careful when measuring SDS because it can aerosolize and
cause coughing fits.
3. APS is an initiator for the polymerization reaction and may
need to be made freshly as this solution can go bad. Therefore,
it is recommended to make only small volume solutions that
will be depleted over shorter periods of times (within the
month), such as 100 mL.
4. SDS can precipitate while mixing the 5× nonreducing loading
buffer solution as it will become quite thick viscous. It is easier
to mix this in a 50 mL conical tube to ensure proper mixing,
then to aliquot into 500–1000 μL tubes and store at −20 °C
until ready to use. Prior to use, warm each aliquot to 37 °C to
be sure to redissolve any SDS that might precipitate out of
solution while being frozen.
5. Normalizing protein concentrations is important to ensure
loading of equal amounts of protein into each individual well
of the polyacrylamide gel. Then, when the signal is developed
at the end of the protocol, it is fair to compare the intensity of
the cleared white bands among the different conditions being
tested by that experiment. If one gel is overloaded with a
larger amount of protein and there is a greater signal, then it
could be interpreted not that the treatment induced greater
amounts of active cathepsins, but that more cathepsins were
loaded into the gel. When testing for amount of active cathepsins
in secreted/conditioned media, equal volumes are usually a
more fair way to normalize the signals since under different
treatment conditions, cells may secrete more or less protein
which would nullify loading gels based on total protein
concentrations.
6. Protein ladders are important for proper determination of
cathepsin identities at the end of the assay. To ensure its visibility
after the Coomassie staining, we recommend loading at least
10–15 μL of protein ladder. Positive controls—samples loaded
that ensure cathepsin activity, visible in the completed zymo-
gram—are important. If there is no signal in the samples that
were loaded, it could just be that buffers were not properly
made, not necessarily that there were no active cathepsins in
the sample. Recombinant cathepsins can be purchased and
loaded into the positive control lane, but this can become
costly over time. A much more inexpensive alternative is to use
extract from RAW264.7 cells, a murine macrophage cell line
that produces an abundant amount of active cathepsins. Then
only 2–4 μg of RAW macrophage cell extract should be loaded
to provide a sufficiently detectable signal that will not over-
saturate the zymogram. Also, with careful loading, be sure the
positive control does not overflow into neighboring wells as it
will produce a signal in one of the experimental samples that
may not be from that sample. For recombinant enzymes, we
recommend loading about 10–20 ng of mature cathepsins to
provide sufficient signal. Cathepsin L requires at least 50 ng
for sufficient signal.
7. By 200 V, it will take about 1 h to run when using the dimensions
described for the gel. However, there can be a loss of resolution
if trying to distinguish intracellular cathepsin K from cathepsin
V. Cathepsin K separates to about 37 kDa by molecular ladder
and cathepsin V is 35 kDa, so there is an important distinction
that can be better visualized by running the electrophoresis at
100 V for 2 h. When cathepsin K is secreted into the condi-
tioned media, it actually runs at much higher molecular size,
most likely due to differential processing/tagging that targets it
for secretion. This is shown in Fig. 2 where A is a zymogram
from conditioned medium from either macrophages or osteo-
clasts and cathepsin K is running much higher (around 75 kDa)
compared to its location at 37 kDa, just above the 35 kDa
cathepsin V signal inside of endothelial cells stimulated with
tumor necrosis factor alpha (TNFα), which we have shown to
turn on cathepsin K [16, 17].
8. Three washes with renaturing buffer after 10 min incubations
for each is the standard protocol, but there is a faster protocol
that uses only 5 min incubations, but still three washes, and
248 Manu O. Platt
Fig. 2 Multiplex cathepsin zymography signals different cells. (a) Conditioned media
from differentiated macrophages (MF) and osteoclasts (OCL) from peripheral blood
mononuclear cells were collected and tested by zymography. Cathepsin K, V, S,
and L signals are shown with cathepsin K at the high molecular weight of 75 kDa
from conditioned media. (b) Human aortic endothelial cells were cultured in the
absence (control) or presence of 10 ng/mL TNFα for 24 h prior to lysis and loading for
multiplex cathepsin zymography. The appearance of the cathepsin K band at 37 kDa
just above the 35 kDa cathepsin V band is shown by that M-shaped band just above
the other. (P-person; P1, P2, P3 are from samples from three different people)
will provide a sufficient signal [15]. This should be tested first

under your own specific experimental cells or tissue conditions
before becoming routine.
9. DTT must be added freshly at this step. It is the reducing
agent and cathepsins require a reducing environment for the
cysteine at the active site to participate in hydrolysis. By thaw-
ing an aliquot once the renaturing stage begins, it should be
ready to add by the time of incubation in the assay buffer.
10. For the assay buffer incubation step, 25 mL is sufficient based
on the size of the container in which the gel is being incu-
bated. This volume may need to be increased or decreased
depending on the size of the container. Most important is to
use a volume that will cover the gels.
11. We prefer to incubate them in airtight plastic-sealed bags filled
with the assay buffer, then submerged in 37 °C water overnight.
However, if that is not available, it also works with placing gels
in a container that is covered, and not airtight, that is put on
an overnight shaker at 37 °C (could be a bacterial incubator or

a shaker in a warm room). It is not necessary to use a shaker,
but that does provide the best results. For better results, keep
gels incubating in separate containers for the renaturing, assay,
and staining steps. There can be artifacts that affect the final
image when the gels are touching or if diffusion is limited.
12. Incubation can be as few as 4 h, depending on the concentra-
tion of enzymes in the sample, but this needs to be determined
in each case. In most cases, this is an overnight incubation that
will provide sufficient signal. Coomassie staining may not take
the full 1 h to begin to see the signal, but it is recommended
to allow for 1 h of staining at least the first time, and then to
continue to monitor for the appearance of cleared white bands
in the positive control before removing the Coomassie stain
and adding destain solution. Observing the stained gel over a
light box is one way to view the staining progress of the
zymogram.
13. Images can be captured by digital cameras on lightboxes,
scanners, or imaging darkboxes. To protect the electronic
equipment, a transparent plastic sheet protector can be used to
protect the staining/destaining solutions from the gels from
damaging or staining the electronics. Then images can be
adjusted in Photoshop or other imaging software, and some
imaging softwares have their own image optimization algo-
rithms. It is important to invert the image prior to densitom-
etry since the zymogram is a white band on a dark background,
and most densitometry programs are best with dark bands on a
white background. NIH ImageJ is free software available to
quantify the intensity of the white bands to determine the
amount of active cathepsins by one treatment over another. It is
available at http://imagej.nih.gov/ij/download.html.
14.
Important information about data interpretation:
Interpretation of pH 6.0 zymogram bands: cathepsin K
37 kDa, cathepsin V 35 kDa, cathepsin S 25 kDa, and cathep-
sin L at 20 kDa. These are from the intracellular/cell extract
signals. From secreted/conditioned media, these positions
may change: catK 37–50 kDa. For pH 4.0 zymogram bands,
cathepsin L generates three signal bands 27, 25, and 20 kDa,
with a cathepsin V band appearing higher at 35 kDa, and a
slight cathepsin S overlap signal at 26 kDa, just above the main
cathepsin L band (Fig. 3). Recombinant cathepsins being used
provide different ranges of signals between secreted and iso-
lated forms vs. intracellular isolated forms. Electrophoretic
migration distance is based on enzyme size, not necessarily
weight. It is important to remember that each cathepsin con-
tains between 5 to 7 disulfide bonds, but by running a nonre-
ducing SDS-PAGE, these bonds are not broken and therefore
250 Manu O. Platt
Fig. 3 Cathepsin inhibitors can be used to selectively identify active cathepsin

bands. Recombinant cathepsins K, L, and S were loaded for parallel zymograms
at pH 4 which select for cathepsins L and V, where, after electrophoresis, one of
the gels was incubated with the cathepsin L inhibitor Z-FY-DMK, and the other
was incubated only with vehicle controls. Incubation with Z-FYDMK, a selective
inhibitor of cathepsin L, inhibited many of the cathepsin active bands, but did not
block all active cathepsin bands. This suggested that the residually active band
was not cathepsin L or susceptible to inhibition by 5 μM Z-FY-DMK
there is no complete linearization of the proteins of interest.

Glycosylation of the enzymes can affect electrophoretic migra-
tion of cathepsins [3]. This will matter for recombinant
enzymes isolated from E. coli or from eukaryotic cells; also
whether the cathepsins are secreted or intracellular also will
affect the electrophoretic migration distance. Fair compari-
sons of cathepsin activity of different samples loaded in the
same gel can be made, but absolute standards can also be
loaded to fit the quantified densitometry signal and calculate
an actual value to compare across different gels [12, 23].
15. Other notes on optimization: 0.75 mm gel thickness provides
the best signals, but 1 mm gel thickness can work. It is impor-
tant to remember that diffusion of the buffers through the
thickness of the gels helps control renaturation of the proteases
and better equilibration of both pH and ion concentrations.
The fresher the samples are, the better the results. Freeze–thaw
cycles cause loss of cathepsin activity.
16. To confirm identity of cathepsins in your samples under specific
conditions, nonreducing Western blots should be run with
antibody confirmation. The cysteine cathepsins can run at dif-
ferent distances when under reduced vs. nonreduced condi-
tions. It is also possible to incubate small molecule inhibitors
in the assay buffer during the overnight incubation stage of
the zymography to selectively block the signal of one cathepsin

over another (cathepsin L inhibitor: Z-FY-DMK), cathepsin K
inhibitor (Z-L-NHNHCONHNH-LF-Boc); serine protease
inhibitor (PMSF); MMP inhibitor (GM6001); broad cathep-
sin inhibitor E-64. Example of the inhibition of the cathepsin
L signal by 5 μM Z-FY-DMK is shown in Fig. 3 with only one
band remaining after pH 4 incubation.
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macrophages and osteoclasts predicted with Gleason RL Jr (2013) Endothelial dysfunction,
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Chapter 24
Transfer Zymography
Daniel Pan, Karl A. Wilson, and Anna Tan-Wilson
Abstract
The technique described here, transfer zymography, was developed to overcome two limitations of
conventional zymography. When proteolytic enzymes are resolved by nonreducing SDS-PAGE into a
polyacrylamide gel with copolymerized protein substrate, the presence of the protein substrate can result
in anomalous, often slower, migration of the protease and an estimated mass higher than its actual mass.
A further drawback is that the presence of a high background of substrate protein interferes with pro-
teomic analysis of the protease band by excision, tryptic digestion, and LC-MS/MS analysis. In transfer
zymography, the proteolytic enzymes are resolved by conventional nonreducing SDS-PAGE, without pro-
tein substrate in the gel. The proteins in the resolving gel are then electrophoretically transferred to a
receiving gel that contains the protein substrate, by a process similar to western blotting. The receiving gel
is then processed in a manner similar to conventional zymography. SDS is removed by Triton X-100 and
incubated in conditions suitable for the proteolytic activity. After protein staining, followed by destaining,
bands representing regions with active protease are visualized as clear bands in a darkly stained background.
For proteomic analysis, electrophoresis is carried out simultaneously on a second resolving gel, and the
bands corresponding to the clear regions in the receiving gel after zymogram development are excised for
proteomic analysis.
Key words Zymography, Electrophoretic transfer, Proteolytic enzyme, Protease, Proteomics,

Nonreducing SDS-PAGE
1 Introduction
Protein zymography, in which samples containing proteolytic

enzymes are separated on nonreducing SDS-PAGE gels copoly-
merized with the protein substrate, has been very useful for the
detection and characterization of proteolytic enzymes with respect
to pH optimum and mechanistic class [1]. Following removal of
the SDS with Triton X-100 [2] and incubation in buffers that favor
protease activity, protein staining reveals clear bands where the
protein substrate had been digested by active proteolytic enzymes.
In this conventional method, however, the molecular mass of the
protease, determined from the migration distance of the band in the
zymogram, is often inaccurate, an anomaly that has been attributed
253
254 Daniel Pan et al.
Fig. 1 Developed electrophoretic transfer zymogram. Bovine chymotrypsin (80 pg)

on a resolving gel (15% T, 2.6% C) was transferred to a receiving gel (15% T, 2.6% C)
containing 0.025% (w/v) gelatin for protein substrate. The zymogram was devel-
oped in 0.1 M Tris–HCl pH 8, at 37 °C with gentle agitation for 24 h. The dark
background represents undigested protein substrate stained with Coomassie blue.
The clear band marks the region where the protease from the resolving gel had
transferred to the receiving gel, then digested the protein substrate
to the presence of the protein substrate in the gel [3]. Transfer

zymography overcomes this limitation (reviewed in [4]). In this
method, enzymes are first resolved by nonreducing SDS-PAGE in
a gel without protein substrate. This is followed by the electropho-
retic transfer of the proteins on the resolving gel to a receiving gel,
the latter incorporating protein substrate; in a process that is very
similar to protein blotting [5]. This was first applied toward detection
of amylase [6], then adapted and improved for the study of proteo-
lytic enzymes [7]. After the electrophoretic transfer, the receiving
gel is incubated with buffer suitable for protease activity followed
by protein staining. The presence of active proteolytic enzymes is
manifested as clear bands in a dark background (Fig. 1). The
positions of the bands correspond to the migration of the prote-
ases in nonreducing SDS-PAGE, without the anomalies intro-
duced by the incorporation of protein substrate in the resolving
gel. This also allows for identification of the protease [7]. Aliquots
of the same samples are resolved simultaneously on two SDS-
PAGE gels. One gel is used to locate the active proteolytic enzymes
by transfer zymography. Bands corresponding to positions of active
protease are excised from the second gel for proteomic analysis.
Transfer Zymography 255
This chapter includes detailed protocols for preparing the receiving

and resolving gels, the electrophoretic transfer, and development
of the zymogram, including steps required if proteomic analysis is
to be carried out.
2 Materials
All solutions described here should be prepared with reagent-grade

chemicals and purified water (resistance at least 13–16 MΩ cm).
Powder-free gloves should be used.
2.1 Nonreducing The electrophoretic system used is that of Laemmli [8].

SDS-PAGE
1. Acrylamide solution: 40% Acrylamide/Bis solution 37.5:1
Components
(Bio-Rad Laboratories, Hercules, CA). Alternatively, dissolve
38.96 g acrylamide and 1.04 g bis-acrylamide in water to a
final volume of 100 mL. Store at 4 °C protected from light.
Avoid inhalation of dust and contact with skin.
2. Running gel buffer: 1.5 M Tris–HCl, pH 8.8. Store at 4 °C.
3. Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8. Store at 4 °C.
4. SDS solution: 10% (w/v) sodium dodecylsulfate (Bio-Rad
Laboratories). Alternatively, dissolve 100.0 g SDS in water to a
final volume of 1.0 L (avoid foaming). Store at room tempera-
ture. Avoid inhalation of the dust by using care in handling and
by wearing a filter mask.
5. TEMED: (N,N,N′,N′-tetramethylethylenediamine). Store at
room temperature.
6. Ammonium persulfate solution: 10% (w/v) ammonium persul-
fate. Dissolve 0.5 g ammonium persulfate in water to a final
volume of 5.0 mL. Prepare just before using.
7. Electrophoresis tank buffer: 0.025 M Tris base, 0.192 M glycine,
0.1% (w/v) SDS, pH 8.3. Dissolve 12.1 g Tris base and 57.6 g
glycine in water. Add 40.0 mL 10% (w/v) SDS solution,
and adjust with water to a final volume of 4.0 L. Store at 4 °C.
Do not reuse.
8. 2× Laemmli sample buffer (Bio-Rad Laboratories). Do not add
2-mercaptoethanol. Alternatively, mix together 2.5 mL stacking
gel buffer, 4.0 mL SDS solution, 2.0 mL glycerol, and water to
a final volume of 10.0 mL. Add 1.0 mg bromophenol blue and
mix until dissolved. Store at room temperature.
9. Commercial prestained protein standards suitable for the mass
range of the proteases of interest. Store at −20 °C.
2.2 Components 1. Transfer buffer: 0.025 M Tris base, 0.192 M glycine, pH 8.3.
for Electrophoretic Dissolve 6.0 g Tris base and 28.8 g glycine in water to a final
Transfer volume of 2.0 L. Store at 4 °C. Do not reuse.
2.3 Zymography 1. Protein substrate stock solution (see Note 1): In the procedure
Components given here, we use 1.0% (w/v) EIA-grade gelatin (Bio-Rad
Laboratories). Dissolve 0.25 g of the protein substrate in water
to a final volume of 25.0 mL. Heat to dissolve, but do not
boil. Cool to room temperature before using.
2. Triton X-100 solution: 2.5% (v/v) Triton X-100. Dissolve
25 mL Triton X-100 detergent in water to a final volume of
1.0 L. Store at room temperature.
3. Zymogram development buffer (see Note 2): This buffer
would set the conditions for optimum activity of the protease(s)
of interest.
4. Protein stain: 0.5% (w/v) Coomassie Brilliant Blue R-250 in
40% (v/v) methanol, 10% (v/v) acetic acid. Dissolve 5.0 g
Coomassie Brilliant Blue R-250 in 0.4 L methanol, 0.1 L gla-
cial acetic acid, and water to a final volume of 1.0 L. Store at
room temperature in a tightly closed container.
5. Destain solution: 40% (v/v) methanol, 10% (v/v) acetic acid.
Mix 1.6 L methanol, 0.4 L glacial acetic acid, and water to a
final volume of 4.0 L. Store at room temperature in a tightly
closed container.
6. Zymogram storage solution: 5% (v/v) methanol, 7% (v/v)
acetic acid. Mix 5.0 mL methanol, 7.0 mL glacial acetic acid,
and water to a final volume of 100.0 mL. Store at room
temperature in a tightly closed container.
2.4 Electrophoresis 1. Vertical gel electrophoresis apparatus and power supply: This
and Electrophoretic protocol was developed using the Mini-Protean Electrophoresis
Transfer Equipment System (Bio-Rad Laboratories, Hercules, CA).
2. Equipment for casting and loading electrophoresis gels: gel
casting stands, clamps, glass plates, spacers (or glass plates with
integrated spacers), and gel combs to cast 1.5 mm thick gels in
the mini-gel format (8 × 7.3 cm2), 125 mL vacuum flask with
stopper, water aspirator or small diaphragm vacuum pump,
felt-tip pen with water-insoluble ink, 50 mL beaker of water
and glass syringe with needle or other delivery device for layer-
ing water on top of poured gel, 50–100 μL glass micro-syringe
or pipettor with long narrow tips for sample loading, paper
towels, plastic wrap.
3. Tank transfer apparatus for performing western blots: This pro-
tocol was developed using the Mini Trans-Blot Electrophoretic
Transfer Cell (Bio-Rad Laboratories).
4. Equipment for the transfer assembly. Transfer cassette com-
patible with the transfer cell, shallow pan to accommodate the
open cassette, filter paper and fiber pads cut to the size of the
gels.
2.5 Equipment 1. Gel cutter, scalpel.

for Development 2. Plastic boxes with lids that seal. The size should allow for incu-
of Zymograms bation of a mini-gel with shaking.
3. Shaking water bath set at the temperature for zymogram
development and modified to secure the plastic boxes.
2.6 Solution 1. Dilute protein stain: 0.1% (w/v) Coomassie Brilliant Blue
for Proteomic Analysis R-250 in 40% (v/v) methanol, 10% (v/v) acetic acid. Dissolve
0.1 g Coomassie Brilliant Blue R-250 in 40 mL methanol,
10 mL glacial acetic acid, and water to a final volume of
100 mL. Store at room temperature in a tightly closed
container.
3 Methods
3.1 Selecting 1. Select (1) the formulation of the running gel layer of the
Conditions for Transfer resolving gel, (2) the formulation of the receiving gel, and (3)
Zymography the duration of the electrophoretic transfer step (see Note 3).
2. Select the protein substrate to be copolymerized with the
acrylamide in the receiving gel (see Note 1).
3. Select the buffer to be used for development of the zymogram
(see Note 2).
4. Plan the duration and temperature for zymogram develop-
ment in accordance with the amounts of samples to be loaded
in the gel lanes (see Note 4).
3.2 Casting 1. To ensure complete copolymerization of the protein substrate

the Receiving gel with the acrylamide, cast the receiving gel at least 24 h before use.
The materials listed in Subheading 2.1, items 1, 2, 4–6;
Subheading 2.3, item 1, and Subheading 2.4, items 1 and 2 will
be needed. It is advisable to cast at least two gels (see Note 5).
2. Using thoroughly clean, dry, and dust-free glass plates, assemble
the gel mold on the casting stand on a paper towel set on a level
surface. Using a felt-tip laboratory pen with water-insoluble
ink, place a mark on the outside surface of the glass plate 2 mm
from the top edge of the glass plate. The gel solution will be
poured to this mark.
3. Perform the following steps quickly (see Note 6). Work at
room temperature. Wear powder-free gloves. Measure the
appropriate volumes (Table 1) of the acrylamide solution,
running gel buffer, SDS solution, water, and protein substrate
stock solution. Mix with gentle swirling in the vacuum flask.
Stopper the flask, and degas for 30 s to 1 min using a water
aspirator or small diaphragm vacuum pump. While degassing,
swirl contents gently, avoiding foaming. Disconnect the flask
Table 1
Formulation of the receiving gel solution with protein substratea
Total acrylamide monomer

concentrationb
10%(w/v) 12.5%(w/v) 15%(w/v)
Components Volume (mL)

Acrylamide solution 5.00 6.25 7.50
Resolving gel buffer 5.00 5.00 5.00
SDS solution 0.20 0.20 0.20
Water 7.60 6.35 5.10
Protein substrate stock solution 2.00 2.00 2.00
TEMED 0.01 0.01 0.01
Ammonium persulfate solution 0.20 0.20 0.20
Makes 20 mL of gel solution, sufficient for two mini-gels
a
All formulations are with 2.6% cross-linker

b
and add the TEMED and ammonium persulfate solution.

Swirl gently to mix and immediately pour into the gel casting
mold to the level of the felt-tip pen mark. Carefully lower a gel
comb into the gel solution at a slant, slowly leveling out to
avoid trapping of air bubbles (see Note 7). Some overflow of
the gel solution may occur. Avoid skin contact, especially with
the solutions containing acrylamide and bis-acrylamide.
4. Leave the gels undisturbed until the gel is well-polymerized, as
indicated by a very distinct interface between the gel and the
comb. Wrap with damp paper towels and seal tightly with
plastic wrap; store at 4 °C.
3.3 Casting 1. This gel will be poured in two layers—the running gel, then the
the Resolving Gel stacking gel layer. It is advisable to cast two resolving gels (see
(See Note 8) Note 5). Gather together all the materials listed in Subheading
2.1, items 1–6 and Subheading 2.4, items 1 and 2.
2. Use a gel mold of the same size as the one used to cast the
receiving gels. Select the gel comb according to the number of
wells required. After assembling the mold, position the comb
between the glass plates; then using the felt-tip pen, mark the
outer surface of the glass plate 1 cm below the teeth of the comb.
The running gel layer will be poured to this level. Place another
mark 2 mm below the top edge of the glass plate. The stacking
gel layer will be poured to this level. Remove the comb.
3. While casting the resolving gel, wear powder-free gloves. Mix

the components for the running gel layer (see Note 9), follow-
ing the procedure in Subheading 3.2, step 3, but use the solu-
tions and volumes in Table 2 and pour upto the lower mark.
Immediately use the syringe and needle to carefully layer water
several mm deep on top of the gel solution. Avoid mixing the
water with the underlying gel. Leave undisturbed until the gel
has polymerized, as indicated by a distinct line between the
water layer and the gel (~1 h).
4. Remove the water overlay by tilting the gel assembly over a
beaker and using a small piece of filter paper to soak up the
water that clings to the glass plates. Use the solutions and
volumes in Table 3 (see Note 10) to prepare the stacking gel
solution. Mix and degas as before (Subheading 3.2, step 3)
Table 2
Formulation of the running gel portion of the resolving gela
Total acrylamide monomer concentrationb
10%(w/v) 12.5%(w/v) 15%(w/v)
Components Volumen (mL)

Acrylamide solution 5.00 6.25 7.50
Resolving gel buffer 5.00 5.00 5.00
SDS Solution 0.20 0.20 0.20
Water 9.60 8.35 7.10
TEMED 0.01 0.01 0.01
Ammonium persulfate solution 0.20 0.20 0.20
Yields 20 mL of gel solution, sufficient for two mini-gels
a
All formulations are with 2.6% cross-linker

b
Table 3
Formulation of the stacking gel solutiona
Components Volumen (mL)

Acrylamide solution 1.00
Stacking gel buffer 2.50
SDS solution 0.10
Water 6.40
TEMED 0.005
Ammonium persulfate solution 0.050
4% (w/v) total acrylamide monomer, 2.6% cross-linker
a
and pour upto the higher mark. Immediately insert the comb
carefully into the stacking gel solution first on a slant and
straightening out later. If there are trapped air bubbles between
the comb and the gel solution, maneuver the comb to a slanting
position and lower again (see Note 11).
5. Leave undisturbed until polymerization is indicated by a distinct
interface between the comb and the stacking gel layer (~1 h).
Wrap the gels (preferably still on the gel casting stand) with
damp paper towels and plastic wrap. Store at 4 °C.
3.4 Preparation 1. Mix each sample with an equal volume of 2× Laemmli sample
and Loading buffer without 2-mercaptoethanol or other reducing agent.
of Samples These samples should not be heat-treated. Samples should be
on the Resolving Gel kept cold (see Note 12).
2. Prepare prestained molecular mass standards for at least one
lane of each gel according to the manufacturer’s instructions,
using the highest suggested amount. Do not add reducing
agent. Do not heat. If possible, load the standards in wells in
an asymmetric pattern from left to right or mark the glass plate
at the side closest to the leftmost well (see Note 13).
3.5 Electrophoresis 1. This procedure must be carried out at 4 °C.

of the Resolving Gel 2. Set the cold resolving gel into the electrophoresis apparatus.
Carefully remove the comb. Add cold electrophoresis tank
buffer to the top and bottom reservoirs of the apparatus. Use
a Pasteur pipette to gently flush the wells with tank buffer
(see Note 14). Use a micropipette or micro-syringe (see Note 15)
to deliver samples under the buffer in the wells. Add a similar
volume of 1X Laemmli sample buffer to wells without sample.
3. Connect the electrophoresis apparatus to the power supply,
and set the power supply to 87 V. Run at constant voltage
until the bromophenol blue approaches the bottom of the gel
(~4 h) (see Notes 16 and 17). Turn off, then disconnect the
power supply, and retrieve the resolving gel. Follow the equip-
ment manufacturer’s instructions and heed all precautions.
4. While waiting, prepare for zymogram development by bringing
the shaking water bath to the desired temperature and equili-
brate the zymogram development buffer to this temperature.
3.6 Electrophoretic 1. The assembly of the sandwich and the electrophoretic transfer
Transfer is done at 4 °C (see Note 12).
2. Wear powder-free gloves throughout. Use a gel cutter or spat-
ula to separate and discard the stacking gel layer. Carefully
transfer the running gel layer to cold transfer buffer in a plastic
container. Agitate the submerged gel gently on an orbital shaker
for 10 min at 4 °C. Repeat with fresh cold transfer buffer for
another 10 min.
3. Unwrap the receiving gel. In a separate container, gently agitate

the receiving gel in cold transfer buffer (2 × 10 min) at 4 °C.
4. After equilibration, leave both gels submerged in cold transfer
buffer. Also soak, in cold transfer buffer, the fiber pads (see
Note 18) and filter paper sheets that have been cut to the size
of the sandwich. Continue working at 4 °C. Lay the open cas-
sette in a large shallow pan and pour cold transfer buffer to
about 1 cm above the cassette. The components for the sand-
wich will be assembled in a specific order (see Fig. 2). On top
of the part of the cassette that will be nearest to the anode, lay
one wet fiber pad, then one sheet of wet filter paper. Next
position the receiving gel, followed by the resolving gel, align-
ing the lower edges of the two gels (see Notes 19 and 20). Lay
one sheet of wet filter paper on top of the gels, followed by
another fiber pad. After addition of each component, readjust
if needed to release trapped air bubbles (see Note 21), then
add just enough cold transfer buffer to keep the buffer 1 cm
above the assembly. Working carefully so as not to shift the
stacked components, close, and latch the cassette.
5. Continue to work at 4 °C. Position the sandwich in the elec-
trophoretic transfer apparatus. Check to make sure that the
receiving gel is closer to the anode and the resolving gel is
closer to the cathode. Use a stirring motor and magnetic
stirring bar in the tank to keep the transfer buffer circulating.
Set the apparatus to run at constant voltage of 15 V per cm
Fig. 2 Assembly of the electrophoretic transfer sandwich

between electrodes (60 V for the Bio-Rad mini trans-blot

electrophoretic transfer cell) for the predetermined length of
time. The same voltage setting is used whether one or two cas-
settes are set up. When transfer time is reached, turn off the
electric current, disconnect the power supply, and remove the
cassettes. Follow the equipment manufacturer’s instructions
and heed all precautions.
3.7 Zymogram 1. At the end of the planned transfer time, turn off and disconnect
Development the power supply. Open the electrophoretic transfer cassette.
The prestained protein molecular mass standards should be
visible on the receiving gel and not on the resolving gel
(see Note 22). Notch the edge of the receiving gel with a
scalpel to mark the positions of the molecular mass standards
(see Note 23).
2. Immerse the receiving gel in Triton X-100 solution in plastic
container with a tight-fitting lid, using one container for each
gel. With an orbital shaker, gently agitate the containers for
15 min at room temperature. Change to fresh Triton X-100
solution and repeat for another 15 min.
3. Remove the Triton X-100 solution and immerse the gel for
20 min in the temperature-equilibrated zymogram develop-
ment buffer, shaking in the water bath for 20 min. Repeat
with fresh buffer for another 20 min. Replace with fresh buffer
and incubate in the water bath for the predesignated tempera-
ture and time period.
4. Immerse the receiving gel in protein stain. Agitate gently
overnight at room temperature. Follow with destaining solu-
tion, changing periodically at regular intervals until clear bands
appear in the dark background. To be consistent, measure the
volumes of the solutions and maintain the same level of agita-
tion. Scan the zymogram or capture the image with a camera.
The band positions on the receiving gel will be at the same
distances traveled by the proteases in the resolving gel; therefore,
molecular mass can be determined to the accuracy possible on
nonreducing SDS-PAGE (see Note 24). For quantification
of band intensities, set parameters in the image analysis pro-
gram such that the intensities in the region of the clear bands
have higher values than the intensity of the background (see
Note 25). The zymograms can be kept for future reference
in zippered plastic bags with 3–4 mL of zymogram storage
solution at 4 °C.
3.8 Protocol 1. Cast and run two identical resolving gels at the same time in
for Proteomic Analysis the same electrophoresis apparatus.
2. After electrophoresis of the samples (see Note 26), immerse one
of the gels (gel P) in dilute protein stain for proteomic analysis.
Stain overnight. Follow with destaining until dark bands are

visible against a clear background.
3. At the same time, prepare the other resolving gel (gel T) for
electrophoretic transfer of proteins to a receiving gel (gel Z) as
described (Subheading 3.6). Develop the zymogram as
described (Subheading 3.7) to visualize the clear bands of
active protease against a dark background.
4. Lay gel P and the developed gel Z side by side. On gel P, locate
and excise the bands that correspond to the active protease
bands on gel Z, then carry out in-gel trypsin digestion and
LC-MS/MS analysis.
4 Notes
1. The ideal protein substrate in conventional zymography is one

that copolymerizes well with the acrylamide and does not
migrate during the electrophoretic run. If the protein substrate
migrates, the developed zymogram will have zones of light
background stain indicating where the gel has been depleted of
protein substrate and dark background stain where there has
been no net loss of protein substrate [9]. In transfer zymogra-
phy, protein substrate that migrates in the electric field will do
so uniformly out of the gel; therefore, zones with different
protein substrate concentrations do not form. This should allow
for experimentation beyond the use of a heterologous substrate
such as gelatin, to include proteins that serve as actual physio-
logically relevant substrates of the proteolytic enzymes studied.
The concentration of the protein substrate is another parameter
that can be varied. A concentration of 0.1% (w/v) is commonly
used; slight reduction in concentration can expand the lower
limits of detection of protease activity.
2. For the zymogram development buffer, select one that promotes
activity of the proteolytic enzyme(s) of interest. Manipulating
pH allows for the detection of different proteases in the same
sample [10]. An initial study done with traditional enzyme
assays or conventional zymography is recommended to deter-
mine the buffer composition that is optimum for proteolytic
activity.
3. Select the formulation of the running gel layer of the resolv-
ing gel so as to achieve resolution of the proteolytic enzymes
of interest, while also allowing for efficient electrophoretic
transfer of proteins out of the resolving gel. For a given per-
centage of cross-linker [(g bis-acrylamide/(g acrylamide + g
bis-acrylamide)) × 100], e.g., 2.6% C as in the protocol given,
proteins of a given size transfer out more readily with lower
total acrylamide monomer concentration (% T) [7], when gel

pore size is larger [11]. Select the formulation of the receiving
gel and the duration for electrophoretic transfer with a view
toward efficient entry of proteins into the receiving gel, a pro-
cess that is also more rapid with lower % T. This has to be
counter-balanced with the understanding that proteins can
pass through, then out of the receiving gel, a process that is
minimized in gels with higher total acrylamide monomer
concentration. These conditions can be determined by ana-
lyzing the efficiency by which prestained protein mass stan-
dards are transferred. This is illustrated for transfer out of a
resolving gel with the formulation 12.5% T, 2.6% C into
receiving gels that were either of the same formulation or that
were 15% T, 2.6% C (Fig. 3). Band intensities of transferred
proteins of different masses were plotted as a function of
transfer time. To illustrate use of such data, for analysis of
proteolytic enzymes expected to have masses of 25–50 kDa,
we would prepare receiving gels with the formulation 15% T,
2.6% C and set a transfer time of 60 min. If proteolytic
enzymes of interest range in size from 20 to 200 kDa, we
would use two sets of transfer conditions, one for enzymes of
lower mass and another for enzymes of higher mass.
Fig. 3 Relative amounts of proteins of different molecular mass transferred as a

function of protein mass, gel formulation, and transfer time. The resolving gel
was 12.5% T, 2.6% C. The receiving gel was (a)–(c): 12.5% T, 2.6% C; (d)–(f):
15% T, 2.6% C. Molecular masses: (a) and (d): 19 kDa (circle), 26 kDa (square);
(b) and (e): 34 kDa (circle), 50 kDa (square); (c) and (f): 90 kDa (circle), 118 kDa
(square). Adapted from [7] with permission from Elsevier
4. The reaction occurs more rapidly with higher temperature so

long as the protease is not denatured. A longer time period for
zymogram development increases sensitivity so that less abun-
dant proteases can be detected [12]; however, the temperature
and time should be selected such that the band intensities are
linear with respect to the amount of protease present in the
sample under the specific conditions for zymogram development
[13]. The upper limits can be determined by loading increasing
amounts of the same sample in different wells of the resolving
gel, then quantifying the intensities of the band after transfer
and zymogram development (Fig. 4). The lower limit of detec-
tion for transfer zymography compared to conventional
zymography depends upon two factors [7]. The sensitivity is
decreased if the proteolytic enzyme passes through the receiv-
ing gel. On the other hand, the absence of protein substrate in
the resolving gel reduces the loss of protease due to protein
substrate binding, usually manifested as a streak above the band in
conventional zymography. Schedules should also be considered
Fig. 4 Linear relationship between band intensity and amount of protease. (a)
Transfer zymogram showing porcine elastase after 60 min transfer from the
resolving gel (15% T, 2.6% C) to a receiving gel (15% T, 2.6% C) with 0.1% (w/v)
casein. Different amounts of elastase solution (μg) with specific activity of 4 μmol
N-succinyl-Ala-Ala-p-nitroanilide hydrolyzed per min per mg were loaded in
each lane. The receiving gel was incubated for 24 h at 37 °C in 0.1 M Tris–HCl
pH 8.0. (b) Band intensities of the major 22 kDa band in the transfer zymogram
after transfer for 30 and 60 min are plotted vs. amount of elastase loaded on the
lanes. Adapted from [7] with permission from Elsevier
in planning the conditions for zymography development.

The procedure from loading samples onto the resolving gel to
the start of zymogram development takes 6–7 h; therefore, it
would be practical to set conditions so that zymogram devel-
opment occurs overnight.
5. We routinely run two resolving gels and prepare two receiving
gels, in case one or the other gel tears during the assembly of
the transfer sandwich. In the event that two pairs of gels are set
up successfully in the transfer cassette, the second set can serve
as a duplicate or used to vary parameters such as time for transfer
or a condition for zymogram development.
6. To avoid polymerization before the gel solution has been
poured into the mold, gel casting steps must be performed
quickly and without pausing between steps until the gel comb
is in place. This is especially important once TEMED and the
ammonium persulfate solution have been added.
7. The comb minimizes contact of the gel solution with oxygen,
which tends to slow down polymerization; and along with the
damp paper towels and plastic wrap, to prevent desiccation of
the gel before use. A one-tooth comb is preferable; but if not
available, other combs or a water overlay as described in
Subheading 3.3, step 3 can be used. In the latter situation, the
receiving gels must be stored in an upright position. The gels
can be stored for about 2–4 days, so long as there are no signs
of drying out along the edges.
8. Because the resolving gel must be cold before use, and because
the receiving and resolving gels are made using many of the
same solutions and equipment, we routinely cast the receiving
and resolving gels at the same time.
9. Protein substrate stock solution will not be needed for the
resolving gel.
10. Regardless of the total acrylamide monomer concentration
used for the running gel layer, the stacking gel is always 4%
(w/v) in total acrylamide monomer concentration.
11. In addition to preventing slowing down of polymerization
by oxygen, the elimination of air bubbles will ensure that the
bottom of the wells are flat so that the bands will have straight
edges.
12. The samples to be analyzed should be in buffers that are
conducive to keeping the proteolytic enzymes active. If it is
necessary to freeze the samples, it is advisable to determine the
conditions for optimal retention of activity and freeze in aliquots
so that samples undergo only one freeze–thaw cycle. Once
thawed, the solutions should be kept cold to prevent autolysis
or digestion by other proteases in the sample. Furthermore,
because the proteolytic enzymes need to be active for zymogram
development, all steps from loading the samples into a cold

resolving gel, through electrophoresis, and electrophoretic
transfer to a cold receiving gel are carried out at 4 °C.
13. Keeping track of the left → right orientation of the resolving and
receiving gels can be challenging. One way to do this is to always
load the prestained molecular mass standards in the leftmost
well. If it is important to have molecular mass standards at the
two extreme ends, the following steps will make it easy to keep
track of the orientation: Mark the glass plate close to the leftmost
sample with a felt-tip pen. Cut off the lower left hand corner of
the resolving gel after electrophoresis. After the transfer process,
before separating the gels, cut off the corner of the receiving gel
adjacent to the cut corner of the receiving gel.
14. This removes traces of partially polymerized gel and ensures
that the bottom of the wells will be flat. Otherwise, the bands
may have a wavy perimeter.
15. It is easier to control sample loading with a micro-syringe; but
the syringe has to be flushed at least five times with water
between samples.
16. It takes longer to complete the electrophoresis at 4 °C than at
room temperature.
17. Electrophoretic transfer must be carried out immediately after
electrophoresis of the resolving gel. If the operator’s schedule
does not allow that, the voltage could be set lower (30 V) so
that electrophoresis proceeds overnight (~10 h), postponing
electrophoretic transfer and zymography development to the
next day.
18. Transfer cassettes are designed to be used with one gel and a
thin membrane. To accommodate the thickness of the second
gel, we use fiber pads that have been compressed through
repeated previous use.
19. Gels tend to tear when they stick to gloves. To prevent this,
wet the fingertips of the gloves with buffer before handling
the gels.
20. If the tendency of two wet gels to slide past each other makes
assembly of the transfer sandwich too difficult, equilibrate only
the resolving gel in transfer buffer. After setting up the cassette
with fiber pad and filter paper, position the resolving and
receiving gels against each other outside of the buffer in the
pan, then place them simultaneously on top of the filter paper,
making sure that the resolving and receiving gels will end up
closer to the cathode and anode, respectively. Continue
adding filter paper and fiber pad, removing bubbles, and clos-
ing up the cassette. After setting up in the transfer apparatus,
initially set the power supply to run at 110 V. As transfer buffer
passes through the gel that was not equilibrated to transfer
buffer (~5–10 min), reset the voltage to 60 V and run at

constant voltage.
21. Rolling a test tube or gently pressing the side of a gloved
finger along the surface can help to release trapped air bubbles.
Take care not to shift the components in the stack.
22. Protein staining of the resolving gel after electrophoretic

transfer can verify that the proteins have been transferred out.
23. We mark the mass standards with notches on the receiving gel
after transfer because the bands can be difficult to detect
against the dark background of the developed zymogram after
staining.
24. Although the molecular mass determined by transfer zymog-
raphy is more accurate than the mass determined by conven-
tional protein zymography, it should be noted that in order to
preserve protease activity, disulfide bonds on the proteins are
not reduced and the samples are not heat-treated. Under such
conditions, the proteolytic enzymes might not be fully dena-
tured, and thus might not bind SDS to attain a uniform charge
density, resulting in slight deviations from the mass that would
be determined by reducing SDS-PAGE with heat treatment.
25. Interpretation of changes in intensities of bands should be
confined to bands on the same zymogram and bands that
exhibit the same mobility.
26. These samples would contain the protease(s) to be identified
and, ideally, would constitute a partially purified sample with a
minimum amount of other proteins.
References
1. Heussen C, Dowdle EB (1980) Electrophoretic polyacrylamide gels to nitrocellulose sheets:
analysis of plasminogen activators in polyacryl- procedure and some applications. Proc Natl
amide gels containing sodium dodecyl sulfate Acad Sci U S A 76:4350–4354
and copolymerized substrates. Anal Biochem 6. Steup M, Gerbling KP (1983) Multiple forms
102:196–202 of amylase in leaf extracts: electrophoretic
2. Lee LT, Deas JE, Howe C (1978) Removal of transfer of the enzyme forms into amylose-
unbound sodium dodecyl sulfate (SDS) from containing polyacrylamide gels. Anal Biochem
proteins in solution by electrophoresis through 184:96–100
Triton X-100-agarose. J Immunol Methods 7. Pan D, Hill AP, Kashou A, Wilson KA, Tan-
19:69–75 Wilson A (2011) Electrophoretic transfer
3. Hummel KM, Penhelter AR, Gathman AC, Lilly protein zymography. Anal Biochem 411:
WW (1996) Anomalous estimation of protease 277–283
molecular weights using gelatin-containing SDS- 8. Laemmli UK (1970) Cleavage of structural
PAGE. Anal Biochem 233:140–142 proteins during the assembly of the head of
4. Wilkesman J, Kurz L (2012) Advances in zymog- bacteriophage T4. Nature 227:680–685
raphy techniques and patents regarding protease 9. Fernandez-Resa P, Mira E, Quesada A (1995)
analysis. Recent Pat Biotechnol 6:106–114 Enhanced detection of casein zymography of
5. Towbin H, Staehlin T, Gordon J (1979) matrix metalloproteinases. Anal Biochem 224:
Electrophoretic transfer of proteins from 434–435
10. Wilder CL, Park KY, Keegan PM, Platt MO 12. Lantz MS, Ciborowski P (1994) Zymographic
(2011) Manipulating substrate and pH in techniques for detection and characterization
zymography protocols selectively distinguishes of microbial proteases. Methods Enzymol
cathepsins K, L, S, and V activity in cells and 235:563–594
tissues. Arch Biochem Biophys 516:52–57 13. Kleiner DE, Stetler-Stevenson WG (1994)
11. Chrambach A, Rodbard D (1971) Quantitative zymography: detection of pico-
Polyacrylamide gel electrophoresis. Science gram quantities of gelatinases. Anal Biochem
172:440–451 218:325–329
Chapter 25
Sequential Detection of Thermophilic Lipase

and Protease by Zymography
Liliana Kurz, Zully Hernández, Lellys M. Contreras, and Jeff Wilkesman
Abstract
Lipase and protease present in cell-free fractions of thermophilic Bacillus sp. cultures were analyzed by
polyacrylamide gel (PAG) electrophoresis. After run, the gel is electrotransferred to another PAG copoly-
merized with glycerol tributyrate, olive oil, and gelatin. This multi-substrate gel was incubated first for
lipase detection, until bands appeared, and then stained with Coomassie for protease detection. Advantages
of this sequential procedure are the detection of two different enzyme activities on a single PAG, beside
time and resource saving.
Key words Lipase, Protease, Sequential zymography, Thermophiles
1 Introduction
When performing zymography, normally, only one substrate is

copolymerized in the gel-matrix for the electrophoresis. Because of
the different steps involved in sample treatment when submitted to
electrophoresis, the enzyme must be reactivated afterwards, gener-
ally by incubating the gel in a specific buffer. The enzyme will then
degrade the copolymerized substrate, and depending on the catalytic
reaction executed by the enzyme, a specific staining method must be
applied to visualize the process. Here, we focus on two types of
hydrolysis. In one hand, we have lipases/esterases (EC 3.1.1.x),
which when assayed under proper incubation conditions, bands are
seen directly on the gel [1, 2]. On the other hand, for proteases
(EC 3.4.x.x), translucent bands are visualized under a deep blue
background when staining with Coomassie blue [3]. The detection
of these enzyme activities has been commonly performed separately.
The method described here allows to detect both enzyme activities
in only one electrophoresis gel [4]. Therefore, the enzymatic activities
to be tested are submitted to polyacrylamide gel electrophoresis
(PAGE) and then the gel is electrotransferred to a different
polyacrylamide gel, containing the substrates.
271
272 Liliana Kurz et al.
As substrate, a mixture of glycerol tributyrate, olive oil, and

gelatin is used for the sequential detection of lipase and protease.
Following electrotransference, the gel is incubated in an activation
buffer containing Tris–HCl and NaCl, until bands are visualized as
clear bands under a grayish background, corresponding to lipolytic
activity. At this stage, the image must be scanned or photographed,
as the next staining step will cover this result. Once Coomassie
blue is applied over the zymogram, proteolytic activity is seen as
clear bands with a deep blue background.
Electrotransference has been used before in PAGE under non-
denaturing conditions for lipase analysis [5]. Enzymes were trans-
ferred to a nitrocellulose membrane and then detected after reaction
with 1-naphtolpalmitate. The use of electrotransference for zymo-
graphical purposes has also been published elsewhere [6].
2 Materials
2.1 Special Reagents 1. Enzyme controls: commercial trypsin (e.g., from porcine
and Equipment pancreas) and lipase.
2. Centrifuge, electrophoresis equipment, electrotransference
unit.
2.2 Sample 1. Sample resuspension buffer: 20 mM Tris–HCl pH 8.

Preparation
2.3 Electrophoresis 1. Resolving gels without substrate: 0.375 M Tris–HCl, pH 8.8,

12% acrylamide/bis-acrylamide (29:1), 0.1% SDS, 0.05%
ammonium persulfate (APS), 0.005% TEMED.
2. Resolving gels with substrate: 0.375 M Tris–HCl, pH 8.8,
12% acrylamide/bis-acrylamide (29:1), 0.1% SDS, tributyrate
2%, olive oil 0.67%, 0.05% ammonium persulfate (APS),
0.005% TEMED.
3. Stacking gels: 0.125 M Tris–HCl pH 6.8, 4% acrylamide/bis-
acrylamide (29:1), 0.1% SDS, 0.05% APS, 0.001% TEMED.
4. Sample buffer under denaturing conditions (1×): 24 mM
Tris–HCl (pH 6.8), 0.8% (w/v) SDS, 10% (v/v) glycerol, and
0.06% (w/v) bromophenol blue.
5. Running buffer without SDS: 25 mM Tris, 192 mM glycine
pH 8.8.
2.4 Silver-Coomassie 1. Fixing solution: 50% (v/v) methanol–10% (v/v) acetic acid.
Staining 2. Coomassie-TCA staining solution: 0.25% (w/v) Coomassie
Blue G-250, 50% (v/v) methanol, 12.5% (v/v) trichloroacetic
acid.
3. Decoloring solution: 50% (v/v) methanol.
Sequential Zymography 273
4. Incubation solution A: 5% (v/v) methanol, 10% (v/v) acetic

acid.
5. Incubation solution B: 30% (v/v) ethanol.
6. Silver solution: 0.1% (w/v) silver nitrate (fresh prepared).
7. Developing solution: 3.75% (w/v) sodium carbonate, 0.03%
(v/v) formaldehyde (fresh prepared).
8. Stop solution: 1% (v/v) acetic acid.
2.5 Electro- 1. Electrotransference buffer: same running buffer without

transference SDS: 25 mM Tris, 192 mM glycine, final pH 8.8 (without
adjustment).
2.6 Zymography 1. Activation buffer: Tris–HCl 50 mM, NaCl 25 mM, pH 8.8.

2. Coomassie staining solution: 0.1% (w/v) Coomassie G-250,
45% (v/v) methanol, 10% (v/v) acetic acid.
3. Destaining solution: 45% (v/v) methanol, 10% (v/v) acetic
acid.
3 Methods
3.1 Biological 1. Prepare a protein extract containing the lipase and protease
Material activity (see Note 1).
2. In the case of bacterial thermophilic cultures, incubate at
55 °C with constant agitation.
3. After maximal protein expression is achieved (see Note 2),
samples are centrifuged at 9500 × g for 30 min at 4 °C.
4. Supernatant is decanted and saved, while pellet is resuspended
in 2 mL 20 mM Tris–HCl pH 8, then sonicated and centrifuged
at 8160 × g for 30 min.
5. Supernatants are ten times heat-concentrated at 60 °C. All
fractions are kept at 4 °C until assay.
6. As controls, commercial trypsin from porcine pancreas and
lipase can be used.
3.2 Electrophoresis 1. PAGE was performed according to Laemmli [7]. Table 1

shows the amounts used for the resolving and stacking gels
enough to prepare one gel.
2. Commercial lipase (~5 μg) and protease (~3 μg) are dissolved
separately in sample buffer under denaturing conditions.
3. Micrograms amounts of cell-free extracts from the thermophilic
cultures were assayed under same conditions.
4. Two 12% gels are run at 100 V for 90 min at 4 °C (see Note 3).
Table 1
Volumes (mL) for preparation of resolving and stacking gel
Solution 12% Resolving gel 4.5% Stacking gel

Distilled water
1.5 M Tris–HCl pH 8.8 1.00 –
0.5 M Tris–HCl pH 6.8 – 0.50
Acrylamide-bisacrylamide solution 1.60 0.30
APS solution 0.04 0.015
TEMED 0.01 0.005
5. After the run, one gel is stained by the silver-Coomassie

protocol (see Note 4). The other gel is submitted to
electrotransference.
3.3 Silver-Coomassie 1. The Silver-Coomassie staining method according to De Moreno

Staining et al. [8] was used (see Note 4).
2. Place gel in fixing solution for at least 30 min. Discard solution
appropriately.
3. Now incubate in Coomassie-TCA staining solution for at least
1 h with gentle shaking. Discard solution appropriately.
4. Wash three times with decoloring solution, 15 min each.
Discard solution appropriately.
5. Add incubation solution A and shake gently for 15 min.
6. Add incubation solution B and shake gently for 15 min.
7. Wash four times with ddH2O, 5 min each.
8. Add silver solution, place in the dark and leave still for 45 min.
Remove solution and discard appropriately.
9. Rinse briefly with water (~15 s).
10. Add developing solution and shake gently until bands appear
(generally 5–10 min).
11. Stop reaction by adding stop solution.
12. Gel may be kept in stop solution until its final destination
(scanning, drying, or discarding).
3.4 Electro- 1. Prepare the substrate gel. The first ingredients of the mix-
transference ture—glycerol, olive oil, water, and acrylamide solution—are
first sonicated (5 pulses, 18% amplitude) and then gelatin,
APS, and TEMED are added (Table 2).
Table 2
Preparation of the substrate mixture gel for zymography (12%)
Component Volume (mL)

Water (dd) 2.7
Acrylamide/bis-acrylamide solution 2.4
Glycerol tributyrate 0.12
Olive oil 0.04
Sonication
2% Gelatin 0.700
10% APS 0.060
TEMED 0.003
Fig. 1 General scheme for the electrotransference setup. (A) Cellulose paper
sheet. (B) Mixed substrate PAG. (C) PAG containing the enzyme activities to be
tested, previously run. Gel (C) is not stained. Protein bands seen is only in the
case a prestained standard was used. Note direction of the current from the
negative electrode towards the positive electrode. Optimal electrotransference
times oscilate between 15 and 30 min
2. Gel is cast correspondingly and assembled into the sandwich

(Fig. 1, see Note 5).
3. The proteins contained in the electrophoretic gels are ready to
be electrotransferred to the substrate gel (containing the sub-
strate mixture). Set equipment at 15 V to electrotransfere for
25 min at 4 °C, using the same running buffer (see Note 5).
3.5 Zymography 1. After transference, incubate the substrate gel for 24 h at 55 °C
in activation buffer (Tris–HCl 50 mM, NaCl 25 mM, pH 8.8)
(see Note 6).
Fig. 2 Zymogram (12%) by electrotransference using glycerol tributyrate, olive oil,

and gelatin as substrates. Thermophilic strains used as sample were culture under
different media, and intra- and extracelullar fractions were tested. Lanes: (1) intra-
cellular fraction [medium 1] (106 μg); (2) extracellular fraction [medium 1] (35 μg);
(3) extracellular fraction [medium 2] (35 μg); (4) extracellular fraction [medium 3]
(5 μg). Sequential detection of the enzymatic activities was performed First (a–d),
lipase activity was visualized after: (a) 2 h, (b) 3 h; (c) 21 h; and (d) 24 h incubation
in activation buffer. (e) Finally, the gel was stained with Coomassie for protease
detection
2. Lipase activity is monitored for 24 h until optimal translucent

bands under a grayish background are seen (see Note 7).
3. At this point, it is strongly suggested to capture an image of
the gel (photograph or scan), as the next step will cover this
result (Fig. 2a–d).
4. Finally, stain the gel with Coomassie staining solution. Leave
overnight for best results.
5. Wash out excess of stain with destaining solution until translu-
cent bands corresponding to proteolytic activity are visualized
under a deep blue background (Fig. 2e).
4 Notes
1. In our case, strains from thermophilic Bacillus sp. were used.

It is strongly recommended to test for enzyme activity with an
alternative method (photometry, fluorescence spectroscopy,
etc.) before performing zymography. SDS is reported to be a
lipase inhibitor [9]. Its presence must be separately tested over
lipase activity.
2. Culture time must be previously determined, in order to
determine when maximal growth and/or maximal protein
expression occurs.
3. Voltage and times used vary among the literature. The best is
to run control experiments in order to determine the best
parameters. We have found that by fixing voltage at 100 V and
placing the electrophoresis chamber inside the fridge, the run
took 90 min until the blue front reached less than 1 cm before
the end of the gel. Please notice that electrophoresis is done

under nonreducing conditions, i.e., 2-mercaptoethanol is
absent, and samples are not boiled. However, denaturing
conditions prevail, as SDS is present in the sample and run-
ning buffer.
4. Many other staining techniques are published [10]. The one
reported here will not be used anymore in our laboratory due
to ecological reasons. Methanol and formaldehyde are toxic
for the environment. Its use must be avoided when possible.
5. Consult manufacturer’s indication according to the model
available in your lab. It is recommended to use 15–20 V, for
15–30 min. Optimal conditions must be tested separately.
6. Incubation time may vary. Bands can be seen already after 2 h
incubation (Fig. 2).
7. If band visualization is not possible under the naked eye, use a
UV lamp as illumination source.
Acknowledgments
This work was supported by the Consejo de Desarrollo Científico

y Humanístico of the University of Carabobo (CDCH-UAC
373-2010) and was benefited by the Venezuelan Law of Science
and Technology (LOCTI) and the Researcher Stimulation
Program (PEII).
References
1. Kwon MA, Kim HS, Hahm DH, Song JK 6. Pan D, Hill AP, Kashou A, Wilson KA, Tan-
(2011) Synthesis activity-based zymography Wilson A (2011) Electrophoretic transfer
for detection of lipases and esterases. Biotechnol protein Zymography. Anal Biochem 411:
Lett 33:741–746 277–283
2. Singh R, Gupta N, Goswami VK, Gupta R 7. Laemmli UK (1970) Cleavage of structural
(2006) A simple activity staining protocol for proteins during the assembly of the head of
lipases and esterases. Appl Microbiol Biotechnol bacteriophage T4. Nature 227:680–685
70:679–682 8. De Moreno MR, Smith JF, Smith RV (1985)
3. Wilkesman J, Kurz L (2009) Protease analysis Silver staining of proteins in polyacrylamide
by zymography: a review on techniques and gels: increased sensitivity through a combined
patents. Recent Pat Biotechnol 3:175–184 Coomassie blue-silver stain procedure. Anal
4. Wilkesman J, Hernández Z, Fernández M, Biochem 151:466–470
Contreras LM, Kurz L (2014) Enhancement 9. Kim MH, Kim HK, Lee JK, Park SY, Oh TK
of sequential zymography technique for the (2000) Thermostable lipase of Bacillus stearo-
detection of thermophilic lipases and proteases. thermophilus: high-level production, purifica-
Amino Acids 46(5):1409–1413 tion, and calcium-dependent thermostability.
5. Brahimi-Horn MC, Guglielmino ML, Gaal AM, Biosci Biotechnol Biochem 64:280–286
Sparrow LG (1991) Nondenaturing protein 10. Manchenko GP (2003) Handbook of detection
electrotransfer of the esterase activity of lipolytic of enzymes on electrophorectic gels, 2nd edn.
preparations. Anal Biochem 196:76–79 CRC Press LLC, Boca Raton, FL
Chapter 26
Calpain Zymography: General Methodology and Protocol

Kevin K.W. Wang
Abstract
Casein zymography has become one of the gold standard assays for monitoring mammalian calcium-activated
proteases (calpains) in purified enzyme, cell, or tissue samples. This calpain zymography method takes
advantages of (1) casein is an excellent substrate for major isoforms of calpains (Calpain-1, 2 and 3), (2)
the embedded casein is digested into small peptides where the calpain bands are located, thus creating a
clear zone upon Commassie blue gel staining, and (3) the calpain isoforms have different gel mobility
under native gel conditions. Casein zymography is also useful in studying reversibility of inhibitor binding
to calpains.
Key words Calpain, Caseinolysis, Protease substrates
1 Introduction
Gelatin zymography was first developed as a sensitive assay for

matrix metalloproteases (MMP) such as MMP-2, and MMP-9,
often in a biological sample [1–3]. Samples are run on denaturing
SDS-polyacrylamide gel and the MMPs are renatured by removal
of SDS from the gel, followed by incubation of a MMP activation
buffer. Calpains are emerging as prominent calcium-activated cyto-
solic proteases in muscle, brain, and other tissues since 1980. There
are at least 14 isoforms of calpain identified, with calpain-1 and
calpain-2 being the ubiquitously expressed isoforms; however, at
the time, similar zymography assays were not available for studying
calpains initially. This is mainly because (1) gelatin is not a good
substrate for calpain, and (2) the two major calpains (calpain-1 and
calpain-2) exist as functional enzyme only as a heterodimer by asso-
ciating with its small subunit (capns1 or capns2), thus denaturing
SDS-gel did not adequately preserve calpain activity.
We thus developed a calpain zymography method by substitut-
ing gelatin with major milk protein casein as the latter is a preferred
substrate for calpains [4]. We also adopted native (non-denaturing)
gel electrophoresis conditions to keep the calpain heterodimer
279
280 Kevin K.W. Wang
together and to avoid denaturing calpains. The gels are incubated

with buffer containing reducing agent (e.g., DTT) and calcium
chloride. Finally, the gels are developed by staining with Coomassie
Brilliant blue G-250. The lightly stained or nonstained gel bands
(clear zones) over a blue-stained background indicate active calpain
bands in the samples.
2 Materials
2.1 Stacking Gel To prepare 5 mL for four gels (For casting your own gels only): 4%
Solution (w/v) acrylarmide solution, 0.10% (w/v) N,N′-methylene-
bisacrylamide, 330 mM Tris–HCl (pH 6.8).
2.2 Separating Gel Make 25 mL for four gels (For casting your own gels only): 12%
Solution (w/v) acrylamide, 0.32% (w/v) N,N′-methylene-bisacrylamide,
375 mM Tris–HCI (pH 8.8), 0.2% (w/v) casein, 3.5% glycerol.
2.3 Non-denaturing 20% (v/v) glycerol, 2 mM 2-mercaptoethanol, 0.004% (w/v)

4× Sample Buffer bromophenol blue, 200 mM Tris–HCI (pH 7.0).
2.4 Non-denaturing 25 mM Tris-base, 192 mM glycine, 1 mM EGTA, 1 mM DTT.
Gel Running Buffer
(pH 8.3)
2.5 Calpain 20 mM Tris–HCl (pH 7.4), 10 mM DTT, 4 mM CaCl2. Keep at
Reactivation Buffer C room temperature.
2.6 Fixing/ Methanol/water/acetic acid (5:4:1).

Destaining Solution
2.7 Staining Solution 0.25% (w/v) Coomassie blue R-250 in Fixing/Destaining solution.
2.8 Special Reagents The following items must be available to perform the experiments:
and Equipments
1. Bovine casein (sodium salt).
2. Calpain 1, human.
3. Calpain 2, native bovine (CAPN2-350B).
4. E64c (irreversible calpain inhibitor).
5. SJA6017 (reversible, calpain inhibitor) [Calpain Inhibitor VI
(CAS 190274-53-4) (sc-293,979)].
6. PD150606 (reversible, calpain inhibitor).
7. Calcium chloride (CaCl2).
8. Bolt® Empty Mini Gel Cassettes (NW2010).
9. Bolt® Empty Mini Gel Cassette Combs, 10-well (NW3010).
Calpain Zymography 281
10. Precast Novex™ 12% Zymogram (Casein) Protein Gels,

1.0 mm, 12-well (EC64052BOX).
11. Xcell SureLock Mini-Cell electrophoresis unit (EI0002).
12. PowerEase® 300 W Power Supply (115 VAC) (PS0300).
3 Methods
3.1 Zymogram 1. To initiate polymerization, ammonium persulfate (0.04%, w/v)

Preparation and TEMED (0.028%, v/v) are added to the separating casein
gel solution.
2. The separating casein gel solution mixture is immediately
poured into the empty gel cassettes and allowed to polymerize
for 30–60 min.
3. After the well-forming combs are inserted into the top of the
cassettes, ammonium persulfate (0.08%, w/v) and TEMED
(0.028%, v/v) are added to the stacking gel solution.
4. The top part of the cassettes is filled with the stacking gel solution
mixture. The gel is allowed to polymerize for 15–30 min.
Alternative: Commercially available precast casein polyacryl-
amide gels can be used (see Note 1).
3.2 Calpain- 1. Calpain-containing samples in 50 mM Tris–HCl pH 7.4,

Containing Sample 1 mM DTT, l mM EGTA is either untreated or incubated in
Preparation 50 mM Tris–HCl (pH 7.4), 3 mM DTT, 2 mM CaCl2 in the
presence of a calpain inhibitor (30 μM, if desired) for 10 min
on ice (final volume 32 μL) (see Note 2).
2. Examples of calpain inhibitors (if used) are E64c (irreversible
calpain inhibitor), SJA6017 (reversible, calpain inhibitor), and
PD150606 reversible, calpain inhibitor).
3. Three microliters of 100 mM EGTA is added to chelate the
calcium ion (total volume 35 μL).
4. Five microliters of the non-SDS-sample buffer is added (total
volume 40 μL).
3.3 Running 1. The casein gel is prerun with the non-SDS running buffer for
of Non-Denaturing 15 min in an ice-water bath. This removes residual polymer-
Casein Gel ization reagents from the gel that might inactivate calpain.
2. Protease-containing samples are then loaded into the wells.
Samples are purified calpain protein (0.5–2 μg), cell lysate
(30 μg protein), or tissue lysate (30 μg) [4, 5] (see Note 3).
3. Rainbow molecular weight markers are also run alongside as
indicators for the progress of electrophoresis.
282 Kevin K.W. Wang
4. Electrophoresis is run at constant voltage (125 V) for about

2 h in an ice-water bath covering at least 50% (in height) of the
gel running unit.
5. The gel is then removed and incubated in Reactivation Buffer
at ambient temperature (21–24 °C) with two buffer changes
of 30 min each.
6. The gel is then further incubated overnight (20–24 h) at
ambient temperature.
3.4 Staining 1. At the end of the proteolysis reaction, incubate the gels in
and Destaining water for 1 h with two changes (15 min each), followed by
of Zymogram 30 min in the fixing solution.
and Calpain Activity 2. The gel is stained with the staining solution for 30 min, fol-
Visualization lowed by the destaining solution with several changes in 2–5 h.
3. Calpain-1 and/or -2 heterodimer will migrate to a distinct
band in the casein gel and upon addition of reactivation buf-
fer, where it will digest the embedded casein. Thus, a clear
zone will be detected over a uniform blue-stained casein back-
ground (Fig. 1).
4. If both calpain-1 and calpain-2 are present in the same sam-
ples, they can be distinguished by their different mobility in
the casein gel (Fig. 1). Calpain-3 can also be detected with this
method (Fig. 1). It is possible to detect other isoforms of cal-
pain (see Note 4).
5. To achieve optimal results, some assay optimization is recom-
mended. In addition, some more sensitive zymogram alterna-
tives are available (see Note 5–8).
Fig. 1 Calpain zymography using casein as embedded substrate. Human calpain

1, human calpain-2, or rat lens calpain-3 isoform LP82 were loaded (2 μg) onto
casein gel, zymogram was developed according to the described protocol.
Position of calpain-1 vs calpain-2 enzymes is shown
6. The gel is stored in 2.5% (v/v) acetic acid until imaging and
quantification using densitometry method (e.g., NIH ImageJ
software).
4 Notes
1. Commercially available precast casein polyacrylamide gels can

be used [Precast Novex™ 12% Zymogram (Casein) Protein
Gels, 1.0 mm, 12-well (Cat. # EC64052BOX, ThermoFisher)].
2. Casein zymogram can be used to distinguish reversible versus
irreversible inhibitor. In this case, inhibitor at optimal concen-
tration (e.g., 30 μM) was preincubated with calpain samples
with 1 mM CaCl2 and 10 mM DTT for 10 min [4]. 5 mM
EDTA is then added to the solution to chelate the calcium.
Samples are then mixed with sample buffer and run onto the
casein gel. Irreversible inhibitor (e.g., E64c) will block calpain
activity in casein gel, while the inhibition of reversible inhibi-
tor (e.g., SJA6017, PD150606) will be lifted with the long
incubation in the reactivation buffer (Fig. 2).
3. Cell or tissue lysate can be prepared by subjecting cell pellet or
pulverized frozen tissue samples to lysis buffer (1% Triton-X100,
20 mM Tris–HCl (pH 7.4), 5 mM EGTA, 1 mM DTT).
Recommended protein concentration is 1–2 mg/mL for the
lysate [4, 5]. The addition of protease inhibitor cocktail should
be avoided. Samples should be freshly prepared before running
for zymogram. If sample storage is needed, 50% by volume of
Fig. 2 Studying reversibility of calpain inhibitors. Reversible calpain inhibitor

SJA6017, PD150606, and irreversible calpain inhibitor E64c (30 μM each) were
preincubated with calpain-2 (2 μg) in the presence of 2 mM calcium chloride and
DTT for 10 min at room temperature before mixing with native gel sample buffer
and loading onto casein gel
284 Kevin K.W. Wang
glycerol should be added and samples stored at −20 °C freezer

for up to 1–3 months.
4. There are at least 14 different isoforms of calpains [6, 7]. Many
of them can potentially proteolyze casein. Thus, casein zymogra-
phy can be used. For example, calpain-3 isoform LP82 and rat
retina [Rt88 (homodimer)] can be detected in the tissue sample
[8, 9]. Calpain-10 is also detectable by casein zymography from
lens tissue lysate from a rat cataractogenesis model.
5. For better sensitivity, FITC- or Fluorescein-labeled casein can be
used, and fluorescence images can be obtained as endpoints [8].
6. Different pH (7.2–7.8) of the reactivation buffer can be used
to enhance the detection of calpain-1 versus calpain 2 in brain
samples after experimental brain injury [5].
7. This assay can also be used to study calpain activation and
subcellular translocation. For example, Zhao et al. [10]
showed that in a rat traumatic brain injury (TBI) study, there
were marked increases in calpain-1 and calpain-2 activity in
cytosolic fraction in the ipsilateral cortex as early as 15 min and
became maximal at 6 h postinjury. In addition, there is also a
shift in calpain-1 activity from the cytosolic fraction to the
membrane fraction occurred at 3 h after injury and became
maximal at 24 h. In contrast, calpain-2 was only detected in
cytosolic fraction.
8. In addition, a variant calpain zymography has been developed
by Dr. Schnellmann’s group using a calpain-preferred fluoro-
genic peptide substrate Succinyl-Leu-Leu-Val-Tyr-7-amino-4-
methylcoumarin (SLLVY-AMC) instead of casein. With this
SLLVY-zymogram method, they were able to find that rat kid-
ney mitochondria contained several calpain-10 splice variants
75 kDa (calpain 10a), 56 kDa (calpain 10c or 10d), and 50 kDa
(calpain 10e) [11].
Acknowledgment
This study is supported in part by NIH grant R21 NS085455-01

(K.K.W.).
References
1. Romanic AM, White RF, Arleth AJ et al (1998) 3. Wang X, Mori T, Jung JC et al (2002)
Matrix metalloproteinase expression increases Secretion of matrix metalloproteinase-2 and -9
after cerebral focal ischemia in rats: inhibition after mechanical trauma injury in rat cortical
of matrix metalloproteinase-9 reduces infarct cultures and involvement of MAP kinase.
size. Stroke 29:1020–1030 J Neurotrauma 19:615–625. doi:10.1089/
2. Wang KKW (2002) Assaying proteases in cel- 089771502753754082
lular environments. Curr Protoc Protein Sci 4. Raser KJ, Posner A, Wang KKW (1995) Casein
Chapter 21:Unit 21.12. doi:10.1002/ zymography: a method to study mu-calpain,
0471140864.ps2112s27
m-calpain, and their inhibitory agents. Arch in young rat lens. Exp Eye Res 67:221–229.
Biochem Biophys 319:211–216 doi:10.1006/exer.1998.0515
5. Zhao X, Newcomb JK, Posmantur RM et al 9. Azuma M, Fukiage C, Higashine M et al
(1998) pH dependency of mu-calpain and (2000) Identification and characterization of a
m-calpain activity assayed by casein zymogra- retina-specific calpain (Rt88) from rat. Curr
phy following traumatic brain injury in the rat. Eye Res 21:710–720
Neurosci Lett 247:53–57 10. Zhao X, Posmantur R, Kampfl A et al (1998)
6. Huang Y, Wang KKW (2001) The calpain fam- Subcellular localization and duration of
ily and human disease. Trends Mol Med μ-calpain and m-calpain activity after traumatic
7:355–362 brain injury in the rat: a casein zymography
7. Sorimachi H, Hata S, Ono Y (2011) Calpain study. J Cereb Blood Flow Metab 18:161–167.
chronicle—an enzyme family under multidisci- doi:10.1097/00004647-199802000-00006
plinary characterization. Proc Jpn Acad Ser B 11. Giguere CJ, Covington MD, Schnellmann RG
Phys Biol Sci 87:287–327 (2008) Mitochondrial calpain 10 activity and
8. Ma H, Shih M, Hata I et al (1998) Protein for expression in the kidney of multiple species.
Lp82 calpain is expressed and enzymatically active Biochem Biophys Res Commun 366:258–262.
doi:10.1016/j.bbrc.2007.11.133
Chapter 27
CTAB Zymography for the Analysis of Aspartic

Proteases from Marine Sponges
Oscar González and Jeff Wilkesman
Abstract
Electrophoresis under denaturing conditions in the presence of SDS is a standard method for the protein
and enzyme scientist. Nevertheless, there are special situations where this method may originate nonoptimal
results. SDS may cause protein aggregation or precipitation. Beyond this, depending on the type of
protein, some just do not resolve well or migrate abnormally in SDS gels. SDS, an anionic detergent, may
be however substituted by a cationic detergent, like CTAB (cetyltrimethylammonium bromide), in order
to solubilize and electrophorize proteins. CTAB electrophoresis allows the separation of proteins based on
molecular weight and can be carried out at neutral or acidic pH. Here, we describe the development of a
CTAB zymography method to analyze aspartic proteases from marine sponges, which present an abnormal
high Rf value when run in SDS-PAGE. The special feature of using CTAB is that it binds proteins, making
them positively charged and thus migrating in the opposite direction compared to SDS-PAGE.
Key words Aspartic proteases, CTAB, Cationic zymography, Electrophoresis
1 Introduction
Aspartic protease analysis is relevant in medicine and biological

science. For example, it is known that HIV presents aspartic protease
activity [1], thus the implementation and enhancement of meth-
ods of higher resolution and effective separation will allow a more
detailed analysis of these enzymes and its inhibition.
Electrophoresis is an analytical method that allows the separa-
tion of biomolecules according to its mass and electric charge
employing a polymeric matrix. There are, however, studies where
atypical molecular weight values of proteases are reported when
using SDS-PAGE [2]. It has become relevant to verify if these
values are valid, and if not, to check the source that originated this
shift in Mr.
Though SDS-PAGE has been a very common choice to perform
electrophoresis, there is an alternative to employ a cationic
detergent—instead of an anionic one like SDS—in an acidic buffer
287
288 Oscar González and Jeff Wilkesman
system, named cationic electrophoresis [3]. This technique is

employed preferentially in the analysis of very acidic proteins that
are not able to interact appropriately with SDS. It is also used in
the analysis of very basic nucleoproteins that behave abnormally in
SDS-PAGE [4]. In this case, the direction of the electric field must
be inverted.
At neutral pH, alkaline proteases as well as very acidic proteases do
not migrate as expected under the electric field when applying electro-
phoresis, resulting in the accumulation of proteins with similar charac-
teristics in the upper part of the resolving gel. This migration pattern is
a hindrance for the protein identification after the separation.
Glycoproteins and lipoproteins also migrate abnormally in SDS-PAGE,
as their non-protein moieties do not uniformly bind the detergent.
Proteins with an unusual amino acid sequence, especially those con-
taining high amounts of Lys, Pro, acidic, or basic residues, are prone to
behave erratically under standard SDS-PAGE conditions [3, 5].
Cationic electrophoresis may result as a more complicated
method compared to SDS-PAGE [4], as polymerization of acidic gels
is somehow more difficult than regular basic gels. As cationic deter-
gent, CTAB has been commonly used [3, 6, 7]. However, the use of
16-BAC is increasing [8–10] (Fig. 1).
A new multiphase system has been described for 16-BAC-PAGE,
in which the phosphate buffer is substituted by acetic acid [11]. This
feature plus the optimization of the 16-BAC concentration and
the use of a new tracking dye allows a highly efficient performance
of the electrophoresis [12]. This leads to a system that is as easy to
manage and is as good—in terms of resolution quality—as a standard
SDS-PAGE.
Here, we have enhanced the cationic electrophoresis by copoly-
merizing gelatin in the gel matrix in order to develop a cationic
zymography method. Zymography is a technique that enables one
to detect enzymatic activity, under nonreducing conditions. Enzyme
Fig. 1 Common detergents used in cationic electrophoresis

CTAB Zymography 289
activity may be inhibited by the presence of the detergent. In this

regard, after the run, the gel is washed with Triton X-100, in order to
remove the detergent and allowing renaturalization of the enzyme.
Afterwards, that gel is placed in an activation buffer, allowing the
enzyme to catalyze hydrolysis reaction of the substrate. The end stage
consists of staining the gel with Coomassie Brilliant Blue (CBB) in
order to visualize the enzyme activity as translucid bands under deep
blue background [13].
2 Materials
2.1 Sample 1. Homogenization buffer: 0.1 M Tris–HCl pH 7.4, 0.05 M

Homogenization NaCl.
2.2 Protein 1. 1 mg/mL BSA solution, Bradford protein determination kit.

Quantitation
2.3 CTAB 1. Resolution gel: 10% acrylamide-bisacrylamide, 60 mM KOH;

Electrophoresis 0.376 M [~2.15% (v/v)] acetic acid buffer pH 4.3.
2. Stacking gel: 5% acrylamide-bisacrylamide, 60 mM KOH,
0.36% (v/v) acetic acid buffer pH 6.7.
3. Sample buffer 2×: 120 mM KOH, 127 mM [~0.725% (v/v)]
acetic acid buffer pH 6.7, 4% (m/v) CTAB, 20% (v/v) glycerol,
0.02% (m/v) methyl green (see Note 1).
4. Running buffer: 350 mM β-alanin, 140 mM acetic acid final
pH 4.5.
5. Activation buffer: 0.1 M acetic acid–sodium acetate pH 4.0.
6. CBB staining solution: 0.25% (m/v) Coomassie brilliant blue
R-250, 40% (v/v) methanol, 10% (v/v) acetic acid.
7. Destaining solution: 40% (v/v) methanol, 10% (v/v) acetic acid.
8. Triton X-100 stock solution 10% (v/v): dilute 10 mL Triton
X-100 until a final volume of 100 mL.
9. Triton X-100 washing solution (1%): dilute 5 mL of Triton
X-100 stock solution (10%) with water until a final volume of
50 mL.
2.4 General Lab Electrophoresis chamber, power supply, ultracentrifuge, photometer,

Equipment ultraturrax.
3 Methods
3.1 Marine Sponge 1. Marine sponge specimens of Amphimedon erina were collected
Homogenate from the Caribbean sea at the Isla Larga, San Esteban National
Park, Carabobo State (10°29′18″N 67°56′40″O).
2. Weigh a 20 g sample of sponge. Cut in small pieces and

homogenize in a mortar with liquid nitrogen and 25 mL
homogenization buffer.
3. Once a soft paste is obtained, homogenize further with an
ultraturrax.
4. Centrifuge the homogenate at 20,000 × g for 30 min at
4 °C [2].
3.2 Quantification 1. Perform total protein determination according to the Bradford

of Total Proteins protocol [14].
2. Prepare a calibration curve employing 1 mg/mL BSA as standard
protein.
3. Read absorbance at 595 nm.
4. Make duplicate of the calibration curve.
3.3 CTAB-PAGE 1. Set up the glasses in order to polymerize the gels. We have
adapted the CTAB-PAGE protocol according to Shi and
Jackowski [15] and to Díaz-López et al. [16].
2. Polymerize the gels according to Table 1.
3. Dissolve sample in sample buffer 2×.
4. Fill the chamber with running buffer.
5. Apply ~0.5 μg of sample per well.
6. Place the lid of the chamber as usual. When connecting the
cables to the power supply, invert the positive and negative plugs.
That is, place the (+) red cable in the black (−) hole of the power
supply, and similarly, place the (−) black cable in the red (+) hole
of the power supply.
7. Begin the run at 60 V for 30 min (at room temperature ~ 28 °C;
see Note 2).
8. Increase the voltage to 120 V and let it run for further 1.5–2 h,
or until methyl green indicator reaches the bottom of the gel
(see Note 3).
9. After run, stain the gel in CBB staining solution. Destain the
excess of dye until bands are visualized.
3.4 CTAB 1. Follow the same steps described in Subheading 3.3.

Zymography 2. For gel polymerization, prepare 10% gels containing 0.1%
gelatine (Table 1).
3. Once the run is finished, place the gel in activation buffer and
incubate overnight (see Note 4).
4. Stain the gel as in step 9 from Subheading 3.3 (Fig. 2).
CTAB Zymography 291
Table 1
Preparation of resolving and stacking gels
Resolving gel Resolving gel Stacking gel

Gel component (10%) (10%) for zymography (4%)
Water 1.735 mL 0.745 mL 1.82 mL
2.15% (v/v) acetic acid—60 mM KOH pH 4.3 2.580 mL 2.58 mL –
0.36% (v/v) acetic acid—60 mM KOH pH 6.7 – – 1.44 mL
1% (m/v) Gelatin – 0.6 mL –
Acrylamide–Bisacrylamide solution (29:1)% 2 mL 2 mL 0.66 mL
10APS 60 μL 60 μL 60 μL
TEMED 15 μL 15 μL 15 μL
Fig. 2 Preliminary result of CTAB electrophoresis

4 Notes
1. Methyl green is included as a track dye, in order to monitor

the run of the gel. Glycerol is included to minimize diffusion
during sample application in the wells.
2. CTAB may precipitate under 15 °C [15]. Thermolabile
enzymes may be an issue. Check first how thermo-resistant
your enzyme is. However, the system may still be placed in a
special refrigerated compartment between 15 and 20 °C.
3. Other positively charged tracking dyes may be used as methy-
lene blue or pyronin Y [16].
4. The exact incubation time must be determined. Sometimes we
obtained bands after 36 h incubation.
Acknowledgments
We acknowledge partial funding from the Consejo de Desarrollo

Científico y Humanístico de la Universidad de Carabobo (CDCH-
2014). We kindly thank the donation of CTAB from Prof. Dr.
C. Cabrele, University of Salzburg, Austria. Sponge samples were
kindly provided by Prof. J.G. Rodríguez, Biology Department,
FACYT, University of Carabobo.
References
1. Brik A, Wong CH (2003) HIV-1 protease: biologically active proteins by cetyltrimethyl-
mechanism and drug discovery. Org Biomol ammonium bromide-polyacrylamide gel elec-
Chem 1:5–14 trophoresis. Anal Biochem 145:170–176
2. Wilkesman J, Schröder HC (2002) Heat-stable 8. Hartinger J, Stenius K, Högemann D, Jahn R
protease from the marine sponge Geodia cydo- (1996) 16-BAC/SDS-PAGE: a two-
nium. Cell Mol Biol (Noisy-le-Grand) dimensional gel electrophoresis system suitable
48:379–383 for the separation of integral membrane pro-
3. Buxbaum E (2012) Cationic electrophoresis. teins. Anal Biochem 240:126–133
Methods Mol Biol 869:55–63 9. Macfarlane DE (1989) Two dimensional
4. Janson JC (2011) Protein purification: princi- benzyldimethyl-n -hexadecylammonium
ples, high resolution methods, and applica- chloride-
sodium dodecyl sulfate preparative
tions. John Wiley & Sons, New York, p 375 polyacrylamide gel electrophoresis: a high
5. Garfin DE (2003) Gel electrophoresis of pro- capacity high resolution technique for the puri-
teins. In: Davey J, Lord M (eds) Essential cell fication of proteins from complex mixtures.
biology, vol 1: Cell structure, a practical Anal Biochem 176:457–463
approach. Oxford University Press, Oxford,
10. Nothwang HG, Schindler J (2009) Two-
UK, pp 197–268 dimensional separation of membrane proteins
6. Eley MH, Burns P, Kannapell CC, Campbell P by 16-BAC-SDS-PAGE. Methods Mol Biol
(1979) Cetyltrimethylammonium bromide 528:269–277
polycrylamide gel electrophoresis: estimation 11. Kramer ML (2006) A new multiphasic buffer sys-
of protein subunit molecular weights using cat- tem for benzyldimethyl-n- hexadecylammonium
ionic detegents. Anal Biochem 92:411–419 chloride polyacrylamide gel electrophoresis of
7. Akin DT, Shapira R, Kinkade JM Jr (1985) proteins providing efficient stacking.
The determination of molecular weights of Electrophoresis 27:347–356
CTAB Zymography 293
12. Braun R, Kinkl N, Beer M, Ueffing M (2007) 15. Shi Q, Jackowski G (1998) One-dimensional
Two-dimensional electrophoresis of membrane polyacrylamide gel electrophoresis. In: Hames
proteins. Anal Bioanal Chem 389:1033–1045 BD (ed) Gel electrophoresis of proteins: a prac-
13. Snoek-van Beurden PA, Von den Hoff JW tical approach, 3rd edn. Oxford University
(2005) Zymographic techniques for the analy- Press, New York, pp 1–52
sis of matrix metalloproteinases and their 16. Díaz-López M, Moyano-López FJ, Alarcón-
inhibitors. Biotechniques 38:73–83 López FJ, García-Carreño FL, Navarrete del
14. Bradford MB (1976) A rapid and sensitive Toro MA (1998) Characterization of fish acid
method for the quantitation of micrograms proteases by substrate-gel electrophoresis.
quantities of protein utilizing the principle of Comp Biochem Physiol B Biochem Mol Biol
protein-dye binding. Anal Biochem 72:248–254 121:369–377
Chapter 28
Zymography Detection of a Bacterial Extracellular

Thermoalkaline Esterase/Lipase Activity
María Tapizquent, Marleny Fernández, Georgina Barreto,
Zully Hernández, Lellys M. Contreras, Liliana Kurz,
and Jeff Wilkesman
Abstract
Lipases are esterases that occur widely in nature, yet those with commercial relevance are exclusively from
microbial origin. Glycerol and long-chain fatty acids are the products after hydrolysis of esters bonds in
saponifiable lipids catalyzed by lipases. In this work, we describe lipase/esterase activity contained in cell-
free fractions from thermophilic bacteria, cultured in medium containing olive oil. Analysis of the cell-free
fractions by electrotransference zymography, using tributyrin as substrate, revealed bands corresponding
to lipase activity. The method is simple, fast, and inexpensive.
Key words Electrophoresis, Electrotransference, Esterase, Lipases, Thermophiles, Zymography
1 Introduction
Lipases are a versatile type of enzymes able to catalyze the hydroly-

sis and—in some cases—the synthesis of triglycerides at the water/
oil surface [1–3]. These multitalented enzymes carry out esterifica-
tions, transesterifications, and a wide range of other reactions [4].
Generally, lipases do not necessarily obey the Michaelis–Menten
model, as catalysis is performed in heterogeneous media [5, 6].
Temperature, organic solvents, and other chemical substances like
detergents (SDS) affect the enzyme structure and its activity. Still,
some lipases are able to conserve their structure and activity after
exposition towards harsh conditions. Using zymography, esterase/
lipase activity can be detected and an average molecular mass deter-
mined. Though some methods have been described, herein we
focus on the electrotransference method for the zymography
accomplishment, as it allows the enzyme to be embedded with
substrate in a separated gel, without altering the Rf value.
295
296 María Tapizquent et al.
2 Materials
2.1 General Electrophoresis chamber, electrotransfer chamber.

Equipment
2.2 Growth Medium Culture medium: 8 g/L nutrient broth (commercial brand)
supplemented with 1% (v/v) olive oil (commercial brand).
2.3 Protein Commercial kits are available. Standard protein solution 1 g/L
Quantification Kit BSA.
2.4 Electrophoresis 1. 12% resolving gels: 0.375 M Tris–HCl, pH 8.8, 12% acryl-
and Zymography amide/bis-acrylamide (29:1), 0.05% ammonium persulfate
Solutions (APS), 0.005% TEMED.
2. 5% stacking gels: 0.125 M Tris–HCl pH 6.8, 5% acrylamide/
bis-acrylamide (29:1), 0.05% APS, 0.001% TEMED.
3. Running buffer: 25 mM Tris–HCl, 192 mM glycine, final
pH 8.0, without SDS.
4. Sample buffer (4×): 125 mM Tris–HCl pH 6.8, 4% (w/v)
SDS without 2-mercaptoethanol.
5. Substrate gel: 12% acrylamide, 2% tributyrin, 0.1% PSA, and
0.05% TEMED.
6. Transference buffer: 25 mM Tris–HCl, 192 mM glycine, final
pH 8.0.
7. Activation buffer: 30 mM Tris–HCl pH 7.4, 200 mM NaCl.
3 Methods
3.1 Bacterial Strains 1. Choose the biological source. Geobacillus sp. isolated from Las
and Culture Conditions Trincheras (Venezuela) hot springs is a Gram positive thermo-
philic strain used in these assays.
2. Culture bacteria in nutrient broth supplemented with 1%
(v/v) olive oil, at 55 °C, and with constant agitation for 192 h.
3. Centrifuge culture at 9500 × g for 30 min.
4. Collect the resulting supernatant (cell-free fraction) and keep
for further analysis.
5. Concentrate the cell-free fraction sample tenfold using a com-
mercial concentrator (Eppendorf) at 60 °C.
6. After concentration, keep samples on ice until further processing
for the gels.
3.2 Protein 1. Perform a protein quantification following the Bradford

Quantification method [7] (see Note 1).
Esterase/Lipase Zymography 297
Table 1
Volumes (mL) for preparation of resolving and stacking gel
Gel component 12% Resolving gel 5% Stacking gel

Distilled water 1.37 1.10
1.5 M Tris-HCl pH 8.8 1.00 –
0.5 M Tris-HCl pH 6.8 – 0.50
30% Acrylamide-bisacrylamide 1.60 0.33
solution
10% APS 0.04 0.015
TEMED 0.01 0.005
2. Build calibration curve with BSA standard protein solution.

3. Measure absorbance at 590 nm.
3.3 Electrophoresis 1. Prepare two gels (12% resolving gel, 5% stacking gel) for the
and Zymography PAGE (Table 1). Take into account that it will be run under
nonreducing (sample buffer without 2-mercaptoethanol and
no heating) and non-denaturing conditions (absence of SDS
in running buffer) (see Note 2). One of the gels will be stained
and the other will be electrotransferred.
2. Prepare the samples by dissolving them in sample buffer (4×).
3. Apply the samples in the wells and run the gel at 100 V for
~90 min on a vertical camera.
4. After the run, take one of the gels and stain it using the silver
staining method [8] (see Note 3).
5. For the zymography process, the remaining gel will be electro-
transferred [9].
6. Rinse the gel with distilled water for 10 min and place it into
the electrotransference set.
7. Previously, a 12% substrate gel must have been casted (Table 2)
(see Note 4).
8. Set the transference sandwich according to the specifications
of the manufacturer (Fig. 1).
9. Carry out the transference in the electrotransference equipment,
at 15 V for 15 min, with transference buffer.
10. After transference, incubate the substrate gel in activation buffer
for 1 h at 55 °C (see Note 5).
11. Activity is visualized as a transparent band in an opaque

background.
Table 2
Preparation of the substrate gel for transfer zymography (12%)
Component Volume (mL)

Water (dd) 1.7
Acrylamide/bis-acrylamide solution 1.2
Glycerol tributyrate 0.06
Olive oil 0.02
Sonication
10%APS 0.030
TEMED 0.003
Fig. 1 Diagram for the electrotransference setup

12. Register the image with a transilluminator or scanner. UV
(280 nm) irradiation may be applied to obtain better gel images,
with a grayish background and dark bands.
13. SDS-PAGE and zymography examples are shown in Fig. 2
(see Note 6).
4 Notes
1. Regard that total protein is being measured (so other proteins

present will be measured as well). In parallel, it is recom-
mended to determine enzyme activity, employing, e.g.,
Esterase/Lipase Zymography 299
Fig. 2 Electropherograms showing lipase/esterase activity present in cell-free

fractions. (a) SDS-PAGE (12%) under nonreducing conditions stained with CBB. (b)
Zymogram (12%) with tributyrin as substrate, accomplished by electrotransfer-
ence. Lanes: (1) Lipase control (9 μg). (2 and 3) Cell-free fractions (~35 μg)
p-nitrophenyl palmitate (p-NPP) as substrate and measuring

absorbance at 420 nm [10–12]. Porcine lipase type II can be
used as positive control.
2. These running conditions assure lipase activity at the end
stage. However, some lipases have been shown to be SDS-
tolerant. In our case, we allow SDS to present in the sample
buffer, but not in the running buffer. This creates a semi non-
denaturing condition in the run that has not been described in
the literature so far.
3. Many staining methods are described [8]. However, you may
choose also the Coomassie staining procedure. The staining
method chosen will depend upon the amount of lipase con-
tained in your sample applied per lane.
4. The electrotransference receptor gel holds the copolymerized
substrate. For our thermophilic lipase, substrates tried were
tributyrin and olive oil. The substrate’s lipophilic nature was
not a disadvantage because copolymerization process allowed
homogeneous distribution in the gel.
5. Timing must be optimized. In our case, bands were possible to
be seen after 1 h incubation. Darker bands were obtained after
24 h incubation [9]. Sometimes activity is seen at room tem-
perature (~25 °C), depending on lipase activity and substrate
affinity. Transparent bands can spread quickly in all the gel.
So, it is recommended to run different enzyme concentrations
to optimize bands revealing.
6. Olive oil may also be used as substrate in the gel. However, we
have seen a preference for tributyrin over olive as substrate.
This reveals that our enzyme is most likely an esterase, according
to reports that define true lipases as those acting only over

long-
chain acylglycerol substrates [13]. Nonetheless, other
descriptions of lipases are less restrictive and define them as
proteins that convert triacylglycerol at the interface between
aqueous and nonaqueous phases [11, 13, 14].
Acknowledgments
This research was partially funded by the Consejo de Desarrollo

Científico y Humanístico (CDCH-UAC-373-2010), University of
Carabobo and by LOCTI. The authors thank the kind assistance of
M.Sc. V. Storaci and D. Quintero.
References
1. Houde A, Kademi A, Leblanc D (2004) Lipases 9. Wilkesman J, Hernández Z, Fernández M,
and their industrial applications: an overview. Contreras LM, Kurz L (2014) Enhancement
Appl Biochem Biotechnol 118:155–170 of sequential zymography technique for the
2. Pandey A, Benjamin S, Soccol C, Nigam P, detection of thermophilic lipases and proteases.
Krieger N, Soccol V (1999) The realm of Amino Acids. doi:10.1007/s00726-014-
microbial lipases in biotechnology. Biotechnol 1707-1
Appl Biochem 29:119–131 10. Sharma R, Chisti Y, Benerjee UC (2001)
3. Jaeger K, Eggert T (2002) Lipases for biotech- Production, purification, characterization, and
nology. Curr Opin Biotechnol 13:390–397 applications of lipases. Biotechnol Adv
4. Gupta R, Gupta N, Rathi N (2004) Bacterial 19:627–662
lipases: an overview of production, purification 11. Nawani N, Kaur J (2000) Purification, charac-
and biochemical properties. Appl Microbiol terization and thermostability of lipase from a
Biotechnol 64:763–781 thermophilic Bacillus sp. J33. Mol Cell
5. Jaeger K, Ransac S, Dijkstra B, Colson C, van Biochem 206:91–96
Heuvel M, Misset O (1994) Bacterial lipases. 12. Salameh M, Wiegel J (2007) Purification and
FEMS Microbiol Rev 15:29–63 characterization of two highly thermophilic
6. Zaks A, Klibanov A (1985) Enzyme-catalyzed alkaline lipases from Thermosyntropha lipolyt-
processes in organic solvents. Proc Natl Acad ica. Appl Environ Microbiol 73:7725–7731
Sci U S A 82:3192–3196 13. Jaeger K, Dijkstra B, Reetz M (1999) Bacterial
7. Bradford MM (1976) A rapid and sensitive biocatalysts: molecular biology, three-
method for the quantitation of microgram dimensional structures, and biotechnological
quantities of protein utilizing the principle of applications of lipases. Annu Rev Microbiol
protein-dye binding. Anal Biochem 72: 53:315–351
248–254 14. Mahadevan GD, Neelagund SE (2013)
8. Chevallet M, Luche S, Rabilloud T (2006) Thermostable lipase from Geobacillus sp. Iso5:
Silver staining of proteins in polyacrylamide bioseparation, characterization and native
gels. Nat Protoc 1(4):1852–1858. structural studies. J Basic Microbiol.
doi:10.1038/nprot.2006.288 doi:10.1002/jobm.201200656
Chapter 29
Amylase Zymography
Adarelys Andrades and Lellys M. Contreras
Abstract
Amylase zymography was carried out for the detection of amylases produced by a Geobacillus stearother-
mophilus strain isolated from the Thermal Center “Las Trincheras” in Venezuela. Zymography is an
electrophoretic technique used to study hydrolases by means of thin gels containing copolymerized-specific
substrates, under nonreducing conditions. In this study, 0.1% starch was incorporated into the gel as
substrate. The formation of clear zones against a dark background in the gel stained with iodine indicated
the presence of amylolytic activity. The thermophilic bacteria released several extracellular amylases to a
selective growth medium supplemented with 1% soluble starch at 55 °C after 40 h incubation. The amylolytic
enzymes showed an optimum temperature of 60 °C and an optimum pH at 6.0. The amylases were par-
tially purified by cold acetone precipitation followed by two chromatographic techniques. These purified
amylases showed different molecular masses which were determined by sodium dodecyl sulfate gel electro-
phoresis and confirmed by zymography.
Key words Zymography, Gel electrophoresis, Hydrolases, Amylases, Thermophiles, Geobacillus

stearothermophilus
1 Introduction
α-Amylases (EC 3.2.1.1) act as starch-degrading enzymes by

catalyzing the hydrolysis of internal α-1,4-O-glycosidic bonds in
polysaccharides with the retention of α-anomeric configuration in
the products. They are mostly metalloenzymes and require calcium
ions for activity, structural integrity, and stability [1]. α-Amylases
can be divided into four basic categories: (1) Endoamylases (cleave
internal α-1,4 bonds resulting in a α-anomeric products), (2)
Exoamylases (cleave α- 1,4 or α-1,6 bonds of the external glucose
residues resulting in α- or β-anomeric products, (3) Debranching
Enzymes (hydrolyze α-1,6 bonds exclusively leaving long linear
polysaccharides), and (4) Transferases (cleave α-1,4 glycosidic
bond of the donor molecule and transfer part of the donor to a
glycosidic acceptor forming a new glycosidic bond) [2–4].
The potential application of this enzyme in industries such as
paper, textile, and food has increased during the last two decades [2].
301
302 Adarelys Andrades and Lellys M. Contreras
As enzymatic liquefaction and saccharification of starch are per-

formed at high temperatures, thermostable amylolytic enzymes are
required and have been currently investigated to improve indus-
trial processes of starch degradation. For example, α-amylases pro-
duced from Bacillus coagulans, B. stearothermophilus, B. caldolyticus,
B. brevis, B. acidocaldarius, and B. thermoamyloliquefaciens are
used extensively for conversion of starch into sugar, syrups and
dextrins, which forms the major part of the starch processing
industry [5–9].
In an attempt to identify new amylases with potential appli-
cation to industry, we have partially purified and characterized
amylases obtained from thermophilic bacteria Geobacillus stearo-
thermophilus, isolated from the Thermal Center “Las Trincheras”
located in Venezuela.
In this chapter, we describe the characterization of several
thermostable amylases by means of a zymographic technique, on
the basis of starch degradation, which is copolymerized in a poly-
acrylamide gel electrophoresis. The bacterial strain was grown in
minimal medium supplemented with 1% starch (see Subheading
2.3), exhibiting a high amylolytic activity after 40–48 h of growth
at 55 °C and pH 7 [10–13]. Cell-free supernatant obtained by
centrifugation was used for estimation of total amylolytic activity,
using the dinitrosalicylic acid (DNS) method [14]. Some proper-
ties related to the effect of temperature and pH on enzyme pro-
duction was also determined. Then, we have partially purified these
amylases employing three conventional methods: (a) cold acetone
precipitation, (b) ion-exchange chromatography, and (c) gel filtra-
tion chromatography. Finally, we have detected the presence of
several amylolytic enzymes on zymograms. Four molecular entities
were identified, with molecular masses corresponding to 64.6,
61.6, 60.3, and 23.4 kDa. Possibly, the association of these in non-
specific complexes was detected in zones of high molecular weight.
2 Materials
All chemicals were of analytical grade.
2.1 Microorganisms Bacterial Geobacillus stearothermophilus strains were isolated from

the Thermal Center “Las Trincheras” (hot springs 55 and 87 °C),
located in central-north Venezuela. The strain with amylolytic
activity was identified as Geobacillus stearothermophilus (Spanish
Type Culture Collection, CECT, Valencia, Spain).
2.2 Stock Solutions Amylase zymography for the determination of amylase activity was
and Reagents performed according to the method described by Laemmli, with
modifications [15]. Zymogram gels are based on 10% polyacrylamide
Amylase Zymography 303
gel electrophoresis which is embedded with 0.1% soluble starch—a

specific substrate for amylases.
1. 1.5 M Tris–HCl, pH 8.8: Weigh 27.23 g Tris base and dissolve
with 80 mL deionized water. Adjust to pH 8.8 with 6 M HCl.
Bring total volume to 150 mL with deionized water. Store at
+4 °C.
2. 0.5 M Tris–HCl, pH 6.8: Weigh 6 g Tris base and dissolve with
60 mL deionized water. Adjust to pH 6.8 with 6 N HCl. Bring
total volume to 100 mL with deionized water. Store at +4 °C.
3. 30% Acrylamide-Bis-acrylamide: Weigh 87.6 g acrylamide,
2.4 g N′N′-bis-methylene-acrylamide. Mix and make up to
volume 300 mL with deionized water. Filter and store at
+4 °C in the dark (see Note 1).
4. 10% SDS: Dissolve 10 g SDS in 90 mL water with gentle stir-
ring and bring to 100 mL with deionized water.
5. 0.1% Soluble starch: Weigh 1 g soluble starch; dissolve in 10 mL
deionized water. Prepare it fresh.
6. 10% APS: Weigh 100 mg ammonium persulfate, dissolve in
1 mL of deionized water. Store the stock solutions in the
freezer at −20 °C.
7. N,N,N′,N′- tetramethylethylenediamine (TEMED).
8. Nonreducing loading sample buffer 4× (without
β-mercaptoethanol) (see Note 2): Mix 1.25 mL 0.5 M Tris
pH 6.8 with 2.0 mL 10% SDS, 2.5 mL glycerol, and 0.2 mL
0.5% bromophenol blue. Add 3.55 mL deionized water to a
total volume 9.5 mL.
9. 10× Running buffer, pH 8.3: Weigh 30.3 g Tris base, 144.0 g
glycine, and 10.0 g SDS, dissolve and bring total volume up to
1000 mL with deionized water. Store at +4 °C. To use: dilute to a
final concentration of 1× running buffer before use (see Note 3).
Generally, we prepare 1 L by mixing 100 mL 10× running buffer
and 900 mL deionized water.
10. Renaturing buffer: 1% (v/v) Triton X-100 in deionized water
(see Note 4).
11. Zymogram development buffer: 50 mM Tris–HCl, pH 7.0,
1 mM CaCl2.
12. KI/I2 Staining solution: Weigh 2 g KI; dissolve in 50 mL
deionized water. Add 1 g iodine and shake it thoroughly to
dissolve. Make up to 100 mL total volume. To use: dissolve
1:10 with deionized water (see Note 5).
13. α-Amylase from barley malt 5.0 mg/mL (commercially available,
used as control).
14. Molecular weight standards, Broad Range (commercially avail-

able): Myosin 200,000 Da, β-galactosidase 116,250 Da, phos-
phorylase b 97,400 Da, serum albumin 66,200 Da, ovalbumin
45,000 Da, carbonic anhydrase 31,000 Da, trypsin inhibitor
21,500 Da, lysosyme 14,400 Da, aprotinin 6,500 Da.
2.3 Medium Geobacillus stearothermophilus strain was cultured in 500 mL

Composition Erlenmeyer flask containing 150 mL of a selective medium con-
for the Production taining 0.2 M phosphate buffered saline (PBS) pH 7.0, 0.00001%
of Amylases CuSO4, 0.00010% FeSO4, 0.00002% MgSO4∙7H2O, 0.00005%
from Thermophiles ZnSO4, 0.00005% NaCl, 0.00010% NH4SO4, 0.00005%
CaCl2∙2H2O, 0.3% yeast extract, 0.3% tryptone, and 1%soluble
starch (all % by mass).
2.4 Equipments Following devices are necessary:

1. Shaker incubator with temperature control.
2. Centrifuge.
3. Concentrator.
4. FPLC—Chromatography system.
5. Electrophoresis system.
6. Imaging System.
3 Methods
3.1 Extraction Geobacillus stearothermophilus strain was grown in selective medium

and Partial Purification supplemented with 1% soluble starch (described in Subheading 2.3).
of Extracellular
1. Incubate 150 mL cultures medium on a shaker incubator
Amylases (70 rpm) at 55 °C for 40 h. Perform colorimetric assays for the
determination of total amylase activity (see Note 6).
2. Harvest 1 L culture with high amylolytic activity and centri-
fuge at 10,000 × g for 15 min at 4 °C. Carefully collect the
cell-free supernatant.
3. Concentrate this supernatant tenfold at 55 °C in glass Petri
dishes (see Note 7).
4. Transfer the tenfold concentrated supernatant to Eppendorf
vials in 1 mL aliquots. Then, add gradually 1 mL of cold ace-
tone (50% final concentration), let it stay for 2 h at 4 °C.
5. Centrifuge at 10,000 × g for 10 min, carefully decant the
supernatant, and keep the pellet (containing amylase activity).
6. Allow the acetone to evaporate at room temperature (~25 °C)
about 15–30 min.
7. Resuspend the pellet in 40 mL 50 mM Tris–HCl pH 7.0 to
obtain the enzyme solution, which is used subsequently for the
purification of the enzyme.
Fig. 1 Amylase zymogram using 0.1% starch as substrate. Active fractions from
an ion-exchange chromatography were analyzed. First lane (left) shows the
α-amylase standard as enzymatic control
8. Perform 10% zymograms to detect amylase activity of the

enzyme solution. A representative zymogram is shown in
Fig. 1.
3.2 Ion-Exchange 1. 5 mL of enzyme solution are diluted with 50 mM Tris–HCl,
Chromatography pH 7.0 to make up 10 mL of total volume.
as First Purification 2. These 10 mL are loaded onto a DEAE column (1.5 × 20 cm),
Step previously equilibrated with 50 mM Tris–HCl pH 7.5.
3. The column is eluted with the same buffer, employing a linear
NaCl gradient from 0 to 1.0 M.
4. The eluate is collected in 1 mL fractions (flow rate 0.2 mL/min).
5. The fractions are concentrated 2.5-fold using a concentrator and
then the amylase activity is detected by zymography (Fig. 1).
3.3 Gel Filtration 1. The active fractions obtained from ion-exchange chromatography
Chromatography are pooled, concentrated 2.5-fold, and then subjected to gel
as Second Purification filtration on a Sephacryl 100 column (0.7 × 30 cm), previously
Step equilibrated with 50 mM Tris–HCl, pH 7.5.
2. The eluate was collected in 0.5 mL fractions (flow rate
0.1 mL/min) using the same chromatographic system as men-
tioned previously.
3. The amylolytic activity of the fractions is also detected by
zymography (Fig. 2).
3.4 Amylases 1. Load the fractions from the different purification steps onto
by Zymography 10% polyacrylamide gels supplemented with 0.1% starch
solution.
Fig. 2 Amylase zymogram using 0.1% starch as substrate. Active fractions from
gel filtration chromatography. First lane (left) shows the α-amylase standard as
enzymatic control
2. Use α-amylase from barley malt (5.0 mg/mL) as a positive

control.
3. Prepare the resolving gel (10% PAGE copolymerized with 0.1%
soluble starch) by mixing 1 mL 1.5 mM Tris–HCl pH 8.8 with
0.400 mL 10% SDS, 1.330 mL 30% Acrylamide/Bisacrylamide,
0.845 mL deionized water and add 0.400 mL soluble starch (1%
stock solution), swirl gently with a vortex. Carefully add 0.020 mL
10% APS followed for 0.005 mL TEMED. Mix thoroughly the
gel solution and carefully pour in between the electrophoretic
glasses. Add a thin layer of deionized water on top of the gel solu-
tion to allow polymerization (see Note 8). Allow 1,5 h for com-
plete polymerization.
4. Prepare stacking gel (4.5%) by mixing 0.500 mL 0.5 M Tris–HCl
pH 6.8 with 0.200 mL 10% SDS, 0.300 mL 30% Acrylamide/
Bisacrylamide, and 0.900 mL deionized water.
5. Now, remove the water layer on the resolving gel by using
filter paper or tissue.
6. Mix gently the polymerizing reagents of the stacking gel with
0.010 mL 10% APS and 0.005 mL TEMED. Carefully pour
the stacking gel on top of the polymerized resolving gel.
7. Insert the comb, trying not to get bubbles stuck underneath
and allow 1 h for complete polymerization.
8. After polymerization, introduce the gel plate into the electro-
phoresis system containing 1× running buffer. Remove
carefully the combs.
9. Prepare the samples by mixing 20 μL sample with 5 μL nonre-
ducing loading sample buffer 4×. Mix by briefly vortexing.
10. If thermostability is going to be additionally tested, submit the

samples to heat (in our case 60 °C for 8 min) (see Note 9).
11. Prepare 5 mg/mL α-amylase standard in nonreducing loading
buffer 4× and include in each zymogram well as positive control
for enzymatic activity.
12. Load 25 μL of the sample per lane and run the gel at constant
voltage (100–150 V), until the bromophenol blue tracking
dye reaches the bottom of the gel (approx. 60–90 min).
13. Remove the zymogram gels from the chamber and place them
in a chamber or Petri plate with 1% Triton X-100 solution for
10 min, with gentle agitation.
14. Briefly, rinse the gels at least five times with deionized water.
15. Incubate zymograms in development buffer for 3 h at room
temperature (~25 °C) on an orbital shaker.
16. Finally, decant the development buffer and stain the zymogram
gels with KI/I2 staining solution until appearance of clear
bands on a uniformly dark background, indicating amylases
activity.
17. Analysis of the amylolytic activity can be performed by an auto-
mated ChemiDoc Imaging System (or similar) in order to visu-
alize the amylase bands on the zymogram gels (Figs. 1 and 2).
18. Molecular mass of the active bands is determined in parallel by
polyacrylamide gel electrophoresis (PAGE) under nonreduc-
ing conditions (see Note 10).
4 Notes
1. Wear gloves and use all safety precautions routinely used when
handling acrylamide solutions.
2. Removing β-mercaptoethanol from the buffer will guarantee
enzyme activity afterwards. Nonreducing loading sample buf-
fer 4× should be prepared fresh just prior to use.
3. 10× Running buffer should not be long-term stored.
Therefore, 1× running buffer should be prepared fresh und
filtered just prior to use.
4. Triton X-100 is used to remove the SDS, allowing enzyme
renaturation. Dissolve 1 mL Triton X-100 in 80 mL water
with gentle stirring and bring to 100 mL with deionized water.
5. The KI/I2 staining solution may stain skin, clothing, or sur-
faces if spilled. The stain is not harmful, but may require sev-
eral days to wear off. Wear protective gloves, protective
clothing, and eye protection. KI/I2 solutions must be pro-
tected from light. Prepare in amber bottles or cover the bottle
with aluminium foil.
6. We have used the classic dinitrosalicylic acid (DNS) method

for the estimation of total amylase activity [14].
7. 1 L supernatant from several simultaneously incubated cultures,
was heat-concentrated at 55 °C to a final volume of 100 mL.
8. Adding water on top of the resolving gel will prevent the con-
tact of the polymerizing gel with the air and keep the gel
straight without any air bubbles.
9. Do not heat the samples above 65 °C, to preserve enzyme
activity.
10. It is suggested to always run in parallel a SDS-PAGE with
molecular weight standards, in order to correlate Rf and
calculate molecular mass of enzymes.
Acknowledgments
This work was supported by Project Pem 2001002268 from the

Venezuelan Fondo Nacional de Ciencia, Tecnología e Innovación.
References
1. Srivastava RAK, Baruah JN (1986) Culture 8. Mielenz JR (1983) Bacillus stearothermophilus

conditions for production of thermostable contains a plasmid-borne gene for α-amylase.
amylase by Bacillus stearothermophilus. Appl Proc Natl Acad Sci U S A 80:5975–5979
Environ Microbiol 52:179–184 9. Hatman P, Wellerson R, Tetrault PA (1955)
2. Aiyer PV (2005) Amylases and their applica- Bacillus stearothermophilus. I. Thermal and pH
tions. Afr J Biotechnol 4:1525–1529 stability of the amylase. Appl Microbiol
3. Zaferanloo B, Bhattacharjee S, Ghorbani MM, 3:7–10
Mahon PJ, Palombo EA (2014) Amylase pro- 10. Freer SN (1993) Purification and characteriza-
duction by Preussia minima, a fungus of endo- tion of the extracellular α-amylase from
phytic origin: optimization of fermentation Streptococcus bovis JB1. Appl Environ Microbiol
conditions and analysis of fungal secretome by 59:1398–1402
LC-MS. BMC Microbiol 14:55 11. Ohdan K, Kurik T, Kaneko H, Shimada J,
4. Shaw JF, Lin FP, Chen SC, Chen HC (1995) Takada T, Fujimoto Z, Mizuno H, Okada S
Purification and properties of an extracellular (1999) Characteristics of two forms of α-amylases
α-amylase from Thermus sp. Bot Bull Acad Sin and structural implication. Appl Environ
36:195–200 Microbiol 65:4652–4658
5. Campbell LL (1955) Purification and properties 12. Najafi MF, Deobagkar D, Deobagkar D (2005)
of an α-amylase from facultative thermophilic Purification and characterization of an extracel-
bacteria. Arch Biochem Biophys 54:154–161 lular α-amylase from Bacillus subtilis AX20.
6. Malhotra R, Noorwez SM, Satyanarayana T Protein Expr Purif 41:349–354
(2000) Production and partial characterization of 13. Pfueller SL, Elliot WH (1969) The extracellu-
thermostable and calcium-independent α-amylase lar α-amylase of Bacillus stearothermophilus.
of an extreme thermophile Bacillus thermooleovo- J Biol Chem 244:48–54
rans NP54. Lett Appl Microbiol 31:378–384 14. Miller GL (1959) Use of dinitrosalicylic acid
7. Egelseer E, Shocher I, Sára M, Sleytr UB (1995) reagent for determination of reducing sugar.
The S-layer from Bacillus stearothermophilus Anal Chem 31:426–428
DSM 2358 functions as an adhesion site for a 15. Laemmli UK (1970) Cleavage of structural
high-molecular-weight amylase. J Bacteriol 177: proteins during the assembly of the head of
1444–1451 bacteriophage T4. Nature 227:680–685
Index
A F
Adenylate kinase��169–178 Fibrin�� 8, 183, 187
Amylase�� 254, 301–308
Aspartic protease��40, 49–51, 137, 287, 292 G
Gelatinase��54, 59, 61, 80, 90–93, 97, 103, 104,
B
116, 147–153, 222–225, 227
Binding mode�� 180, 186 Guaiacol POD��199–204
C I
Calpain�� 25, 26, 279–284 In situ zymography (ISZ)��4, 59, 61–62, 66–67,
Cancer��6, 26, 54, 55, 84, 98, 103, 115, 239, 240 133, 136, 137, 139, 143, 190, 205
Cardiovascular diseases�� 55, 115
Casein L
dye-quenched casein��142 Leishmania�� 214, 215, 218
Catalase��195–197 Leptospira�� 104–106, 110
Cathepsin�� 25–28, 71, 158–160, 163, 164, 239–251 Lipase�� 190, 276, 277, 299
Collagen��8, 71, 74, 75, 80, 93, 97, 103, 115–117, 119,
120, 128, 129, 140, 147, 148, 205, 213, 215–218, 225 M
Collagenase�� 54, 61, 71, 74–77, 80, 103,
Matrix metalloproteinase (MMP)
104, 115–120, 222, 223, 225
MMP-2�� 54–60, 66, 67, 84, 85, 88,
Coomassie brilliant blue (CBB)��14, 16, 18, 19,
91–94, 96, 97, 126, 148, 150, 153, 279
21, 34, 60, 66, 72, 76, 108, 117, 127, 128, 150, 159,
MMP-7�� 54, 59, 60, 68
179, 181, 183–184, 207, 210, 216, 218, 225, 256, 257,
MMP-9�� 54–57, 59, 60, 85, 88, 91–94,
280, 289, 290, 299
96–98, 126, 148–150, 153, 279
Cetyltrimethylammonium bromide (CTAB)��287–292
Microbial hotspots��230
Cysteine protease��26
N
D
Neurodegeneration��54
Drilosphere��229
Neutral zymography��35–38
Duck liver�� 164, 165
P
E
Peroxidase�� 191, 204
Earthworm�� 23–229, 232, 233
Posttranslational modification isoforms��149
Electroblotting��175
Proteases��25–30
Electrophoresis�� 3, 15, 26, 34, 43, 61, 75,
aspartic��29, 33–40, 43–52, 287, 292
85, 104, 116–117, 126, 133, 149, 158, 171–172, 175,
cysteine�� 25–30, 40, 71, 113
176, 179, 189, 201, 205, 213, 223, 239, 255, 271, 279,
metallo��33, 40
287, 296, 302
serine�� 13–23, 26, 29,
Electrophoretic isoforms�� 195, 196
40, 137, 251
Electrotransference/electrophoretic transfer��5, 254–257,
Proteomic�� 6, 254, 255,
260–263, 267, 268, 272–276, 295, 297–299
257, 262–263
Esterase..................................................... 190, 271, 295–300
DOI 10.1007/978-1-4939-7111-4, © Springer Science+Business Media LLC 2017
309
Zymography: Methods and Protocols
310 Index

R Tissue inhibitors of metalloproteinases (TIMPs)��55,
59, 80, 81, 85, 92, 98, 125, 126, 222
RAMA casein zymography��19, 23 Trypanosomatids
Trypanosoma brucei�� 214, 215, 218
S Trypanosoma cruzi�� 214, 215, 218
Safety regulations��9, 142 Two-dimensional electrophoresis (2DE)�� 6, 161–164
Serine protease�� 23, 26, 40, Two-dimensional zymography (2DZ)�� 5, 6, 152,
137, 251 157–165
Silver stain�� 7, 179–187, 297
Y
Sodium dodecyl sulfate (SDS)��15, 27, 35, 44, 56,
72, 85, 104, 126, 149, 159, 170, 179, 200, 205, 214, Yeast��34, 38, 39, 195, 196, 304
224, 240, 253, 272, 279, 287, 295, 303
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Z
(SDS-PAGE)��3, 18, 19, Zymography
26, 29, 33, 72–76, 85, 95, 112, 126, 149–153, 159–161, cationic��288
163–165, 177, 180, 183, 184, 201, 202, 205, 207, 210, in-gel��4, 5, 8, 148, 149
215, 239, 241, 245, 249, 253, 255, 262, 268, 287, 288, in-situ�� 4, 59, 61, 66, 67, 133–143,
298, 299, 308 190, 205
Steatosis��158 in-situ soil��237
Superoxide dismutase (SOD)��189–197 in-vivo��4, 190
multiplex�� 239, 248, 250
T
sequential��271–277
Thermophiles��304 substrate immersing��205–211
transfer�� 5, 6, 253–268, 298

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Methods in

Molecular Biology 1626

For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Library of Congress Control Number: 2017943128

© Springer Science+Business Media LLC 2017

Printed on acid-free paper

This Humana Press imprint is published by Springer Nature

To our children, Jarod and Kylie

To Prof. Ricardo Maldonado, former Rector-President of the University of Carabobo, who

Valencia, Venezuela Jeff Wilkesman

Part II Endopeptidase Zymography

Part III Reverse Zymography and In Situ Zymography

12 Cell In Situ Zymography: Imaging Enzyme–Substrate Interactions . . . . . . . . . 133

Part IV 2D Zymography

Part V Special Zymography Cases

26 Calpain Zymography: General Methodology and Protocol . . . . . . . . . . . . . . . . 279

Adarelys Andrades • University of Leipzig, Leipzig, Germany; Centre for Environmental,

Oscar González • Centre for Environmental, Biology and Chemistry Research, Facultad

Cláudia Jassica Gonçalves Moreno • Programa de Pós-graduação em Bioquímica,

Kanika Sharma • Division of Structural Biology and Bioinformatics, CSIR-Indian

Key words Electrophoresis, Enzymes, Zymography, Troubleshooting

Zymography is the technique based on sodium dodecyl sulfate poly-

Different types of zymography exist, according to the type of

used, as after oxidation they lose their fluorescence. Derivatives of

Zymography allows a number of advantages when analyzing unpu-

As can be seen, zymography provides general and specific and

The choice of a given zymographic method may depend on highly

procedure, chemicals involved, mode of application of the

6. pH conditions: this variable is hardly enough studied. Given

hydrolyzed under inappropriate pH conditions. An inadequate

We would like to thank Prof. Dr. J. Bubis and M. Calabokis

Serine Protease Zymography: Low-Cost, Rapid, and Highly

Serine proteases are known to be one of the major enzymes spread-

To detect serine protease activities, protein substrate zymogra-

2.2 Protease 1. Twenty-five percent of Triton X-100 stock solution is prepared

All procedures are carried out at room temperature.

3.5 Electrophoresis 1. Start electrophoresis without pre-run at constant current

3.7 Incubation 1. After 10 min shaking in an incubation buffer, start incubation

Fig. 1 Comparison of detection sensitivity among three methods such as conven-

4. As example, the detection sensitivity among three zymography

Fig. 2 High sensitive detection of MMP7 (Matrilysin) by RAMA casein zymogra-

Therefore, RAMA casein was revealed to be more sensitive by

1. Acrylamide and bisacrylamide are purchased at the highest

3. This solution should be prepared freshly and from power form,

9. Previously, even in our publication [24], NaCl is contained in

protease band. Protein at a low concentration is unstable and

The author is grateful to Mariko Kawasaki-Yasumitsu for her excel-

Cysteine Protease Zymography: Brief Review

Key words Cysteine protease, Zymography, Cathepsin

Cysteine proteases were formerly known as thiol proteases. This

such as cancer, AIDS, osteoporosis, arthritis, atherosclerosis, as

Cysteine protease Optimal assay

2.1 Protocol 1: 1. 11% polyacrylamide gels containing 0.1% gelatine.

2.2 Protocol 2: 1. Non-reducing loading buffer 5×: 0.05% bromophenol blue,

2.3 Protocol 3: 1. 11% polyacrylamide gels containing 0.1% gelatine.

2.4 Protocol 4: 1. 12.5% SDS–polyacrylamide gels containing 0.2% gelatine.

7. Inhibitors: final assay concentration prepared in activation buf-

Valencia, Venezuela Jeff Wilkesman

in zymography. Avoid heat, detergents, foaming, and over-