Separation of Proteins

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Separation of proteins
Sodium Dodecyl Sulfate–PolyAcrylamide Gel Electrophoresis (SDS PAGE) is an analytical

method used for the separation of charged molecules of proteins in mixtures by their molecular
masses in an electric field. For the segregation of proteins in natural medium there must be a negative
charge on proteins. For this purpose such salts which produce more negative radicles i.e magnesium
sulphate, phosphates and nitrates are used. These negative radicles react with protein molecules and
charged those negative. Most commonly used is SDS and sodium loral sulphate. A reasonable length
of protein molecules (chain of amino acids) is required to move under the influence of electricity
because small size proteins are disappeared. This problem is removed with the addition of glycerol
which prevents the protein form electric shock and density of short chain amino acid is increased.
Uses of SDS PAGE:
• Used to segregate the proteins and nucleic acid.
• By using this we can calculate the number of proteins and can determine their molecular weight
and by the number of proteins and molecular weight we can differentiate/ identify the
microorganisms.
• This is the basic step to perform western blotting.
Electrophoresis is generally of two types:
1. Horizontal gel electrophoresis 2. Vertical gel electrophoresis
In horizontal gel electrophoresis only one chemical is used and pore size is constant throughout
the gel. Movement of electrons is on base of charge to mass ratio.
Vertical gel electrophoresis
In this gel more than one chemical are used to make gel and pore size vary in gel. This gel is
prepared in two layers. Lower layer has more number of molecules in dense form with smaller pore
size and upper layer has less number of molecules in loosely attached form with large pore size. Pore
size gradually decrease from top to bottom. In this gel molecules move under the influence of
electricity and are restricted due to pore size variation. Due to restriction of movement in gel, heat is
produced which can melt the gel. So this should be performed in air conditioned lab. In vertical gel
electrophoresis more time is required which is dependent on thickness of gel and length of gel. SDS-
PAGE is a vertical gel electrophoresis technique.
GEL CONDITIONS:
Electrophoresis can be performed in two conditions depending upon the protein source.
I. Natured condition 2. Denatured condition
Natured /native/naive :( under natural conditions)
Native PAGE is used to separate proteins in their native states according to difference in their
charge density. Native state of protein means proteins are in properly folded state, not denatured or
unfolded state. There are no denaturants present in the gel and buffer in the gel maintains the protein
in its native state. Many proteins are shown to be enzymatically active after separation by native
PAGE. Thus, it is used for preparation of purified and active proteins. Native PAGE can be carried out
near neutral pH to avoid acid or alkaline denaturation to study conformation as well as self-association
or aggregation, and the binding of other proteins or compounds.
When proteins are assembled and bound to each other than sample preparation is needed to
make proteins independent before running in gel. In case of virus confirmation by proteins through
SDS-PAGE (vertical gel electrophoresis) proteins are needed to be denatured due to their assembly in
virus.
Buffer system:
Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain the
pH at a relatively constant value
Two types of the buffer system are used
• Continuous (single buffer system) in this system one type of buffer of same pH is used.
• Discontinuous (Stacking gel & Resolving gel/ double buffer system)
Acrylamide polymerization
The gels typically consist of acrylamide monomers comprising on acrylamide &
bisacrylamide, both having different orientations. The backbone of acrylamide comprised on carbon
molecule that imparts the inert nature of both monomers. First of all these monomers needs to be
reactive and it is possible with the addition of SDS that gives negative charge to both monomers. After
that ammonium per sulfate (APS) is added that positively charged the both monomers, where empty
spaces due to NH4 + ions. A catalyst TEMED is also added to increase the speed of
reaction/polymerization after addition of APS.
Preparation of Resolving gel in Tris /HCl pH 8.6
The polymerization reaction within 15-20 minutes/ 20CO forms a gel because of cross-linking
between two acrylamide molecules. N-Butanol may be added to the resolving gel; butanol removes
bubbles and makes the surface smooth. Then this solution is poured on casting of equipment. Bigger
molecules having smaller pore size moves downward quickly under the influence of gravitational force
than smaller molecules having larger pore size. Thus pattern of gradually decreasing pore size is
created automatically in the resolving gel. Resolving gel is made up of 7-15% concentration to separate
the proteins. Concentration of the gel depends upon the molecular weight of the viral proteins
Protein of 500 kDa (7% of gel is used)
Protein of 300 kDa (10 % gel is used)
Protein of 200 kDa (12 % gel is used,
Protein of 100 kDa (15 % gel is used)
Stacking gel preparation:
After polymerization of resolving gel, stacking gel is poured. Concentration of gel is 2%-5% generally
4% gel is used, pH of the gel is 6.8 (slightly acidic) as of loading buffer. After pouring of stacking gel,
comb is placed. After polymerization, remove the comb by static hands.
Electrophoresis:
Placed the gel in electricity chamber, and add running buffer of pH 8.6
Composition of running buffer
• Tris HCI for pH
• SDS- for provision of -ve charge
• Glycine ~ for straight line movement of proteins vertically
• Chloride ions are present in buffer
Glycine blocks the horizontal movement of proteins and layer of glycerol blocks the reverse
movement of proteins. Heavy proteins travel smaller distance and light proteins covers more distance.
Known molecular weight proteins are also run as standard protein markers.
Molecular weight of the viral proteins should fall within the range of proteins makers e.g In
IBD heavier proteins is of 91-92kDa and lighter protein is of 26-28kDa. Marker for this should contains
a protein of mol. weight higher than 91-92kDa and other proteins lower than 26-28kDa.
Perform electrophoresis in room with low temperature and constant current supply. All the samples
should stack at the same time on resolving gel. If line is not straight then polymerization of gel is not
proper. When electrophoresis is completed then gel is taken out and stoking gel is isolated. Make a
knot on resolving gel at side where samples were stopped.
Staining the gel
Following electrophoresis, the gel may be stained with Coomassie Brilliant Blue or silver stain
to visualize the separated proteins. After staining, different proteins will appear as distinct bands within
the gel according to their sizes (and therefore by molecular weights). The molecular weight of a protein
in the band can be estimated by comparing it with the marker proteins of known molecular weights.
The separated proteins can be cut from the gel and further analyzed by other proteomics techniques.
Staining components:
Coomasei brillent blue---basic dye
Glacial acitic acid--- act as mordent
Methanol ---- act as carrier for dye
De-staining components:
De-staining solutions contain all components of staining solution except the dye.
Rf value vs log molecular weight are used to draw standard curve for the determination of
molecular weight of proteins.

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Sodium Dodecyl Sulfate–PolyAcrylamide Gel Electrophoresis (SDS PAGE) is an analytical

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