Name: Stream: B02

ID Number: Date: September 13, 2023.

Title: Protein Determination Using Bradford Assay

Methodology: The experiment was executed as outlined in the BioC 1021 lab Manual.

Questions:
1. From the information provided, calculate the protein concentration for each sample.
(20 marks)

Using the formula:

C1V1 = C2V2
Where: C1 = initial concentration C2 = final concentration (unknown)
V1 = initial volume V2 = final/total volume
C 1V 1
Transpose for the unknown, ∴ C2 =
V2

Calculations for Sample 1

C 1V 1
Conversion: µm to mm 🠆 C2 =
V2
0.5 mg/ml x 0.010 ml
1000µm = 1mm C2 =
2.0 ml
−3
5x 10
10µm ÷ 1000 C2 =
2.0
= 0.010mm C2 = 2.5 x 10-3 mg/mL

Calculations for Sample 2

C 1V 1
Conversion: µm to mm 🠆 C2 =
V2
0.5 mg/ml x 0.020 ml
1000µm = 1mm C2 =
2.0 ml
−2
1.0 x 1 0
20µm ÷ 1000 C2 =
2.0
= 0.020mm C2 = 5.0 x 10-3 mg/mL

Calculations for Sample 3

C 1V 1
Conversion: µm to mm 🠆 C2 =
V2
0.5 mg/ml x 0.030 ml
1000µm = 1mm C2 =
2. Oml
 −2
1.5 x 1 0
30µm ÷ 1000 C2 =
2.0
= 0.030mm C2 = 7.5 x 10-3 mg/mL

Calculations for Sample 4

C 1V 1
Conversion: µm to mm 🠆 C2 =
V2
0.5 mg/ml x 0.040 ml
1000µm = 1mm C2 =
2. Oml
−2
2.0 x 1 0
40µm ÷ 1000 C2 =
2.0
= 0.040mm C2 = 100.0 x 10-33 mg/mL

Calculations for Sample 5

C 1V 1
Conversion: µm to mm 🠆 C2 =
V2
0.5 mg/ml x 0.050 ml
1000µm = 1mm C2 =
2. Oml
−2
2.5 x 1 0
50µm ÷ 1000 C2 =
2.0
= 0.050mm C2 = 12.5 x 10-33 mg/mL

Calculations for Sample 6

C 1V 1
Conversion: µm to mm 🠆 C2 =
V2
0.5 mg/ml x 0.075 ml
1000µm = 1mm C2 =
2. Oml
−2
3.75 x 1 0
75µm ÷ 1000 C2 =
2.0
= 0.075mm C2 = 18.75 x 10-3 mg/mL

Calculations for Sample 7

C 1V 1
Conversion: µm to mm 🠆 C2 =
V2
0.5 mg/ml x 0.100 ml
1000µm = 1mm C2 =
2. Oml
−2
5.0 x 1 0
100µm ÷ 1000 C2 =
2.0
= 0.100mm C2 = 25.0 x 10-3 mg/mL
 2. Plot all calibration points on a sheet of graph paper. Draw the best smooth line, straight

or curved, through the points. (15 marks)

3. Using your graph and the measured OD at 595 nm, determine the concentration of your

unknown protein. (5 marks)

According to the graph, the concentration of the unknown protein after extrapolation is

19.6 x 10-3 mg/mL.

4. Think about whether or not it should start at the origin and give your reasons. (5 marks)

The best fit curve should start at the origin (0,0) because the blank sample does not have

any of the protein samples included in it. Hence, when placed in the spectrophotometer, an

absorbance of 0.0 OD at 595 nm with a protein concentration of 0.0mg/mL, which would have

been the first recorded and plotted point of the graph. Beer-Lambert’s law is represented by a

linear line with a y-intercept of 0. A = Ɛcl is a representation of y = mx+c. This lab is in

accordance with Beer-Lambert's Law, as it is evident that the absorbance and the concentration

have a directly proportional relationship, with a y-intercept and origin of 0.

5. Define accuracy and reproducibility. (10 marks)

Accuracy can be defined as the degree to which the result of a measurement conforms to

the correct value or a standard and it essentially relates to how close a measurement is to its

agreed value; it is the correctness of a single measurement. Accuracy is ascertained by

comparing the measurement against the true, valid or accepted value. When attempting to attain

results of high degrees of accuracy, precautions need to be adhered to and sources of errors need

to be little to none. In this experiment to determine the concentration of protein in the different

samples, the apparatus used such as the spectrophotometer and micropipette were ensured to be

properly used in order to avoid errors and get accurate results. The disposable pipette tips were

changed when necessary (for example, when touched by surfaces such as other solutions, the

hand, the lab table among others) to avoid contamination of other samples, it was ensured that

light passed through the sample as much as possible by wiping off any smudges that might have

been on the transparent sides of the cuvette and avoiding scratches that would have caused any

deflection. Also, it can be concluded that the results obtained some degree of accuracy as it

reflects the principle of Beer-Lambert’s Law which is defined as the absorbance being directly

proportional to the concentration (c) of the solution and the light path length (l) (Clarke, 2019).

This equation can be represented by a linear line or curve which illustrates direct proportionality

and proves the results gathered after plotting the graph. Finally, the spectrophotometer was

blanked by using a solution that contained water and Bradford reagent and no protein (BSA), so

that when the absorbance of the sample was measured, it is known that the light passing through

is deflected only by the protein and that the absorbance readings only account for the BSA

present. Accuracy also reflects how close a value is to a standard, which is an accepted value.

Accordingly, the results obtained were certainly accurate.

The term reproducibility is associated with the consistency of measurements. It is the

extent to which a tool or equipment can yield the same result when used repeatedly by different

individuals, using the same input variables and methodological steps at various locations under

the same analysis conditions. Reproducibility is pertinent to this experiment because a duplicate
of each protein determination sample was made. This was to see if the absorbance reading of 1B

would be in the same range of values as 1A, and such was observed for most of the samples

because the same conditions and procedures were applied, for example, the incubation time and

the volumes of reagents used were identical and the cleaning of the cuvettes and their appropriate

insertion into the spectrophotometer was recurrent.

Thence, it can be concluded that the results from this experiment were accurate and

reproducible.

6. Name TWO (2) alternate methods that could be used to determine protein concentration

in a sample. In your answer, briefly describe the principle of each method. (10 marks)

In addition to the Bradford Assay, the Ultraviolet Absorption Method and the Lowry

Assay are two additional time-efficient methodologies of determining the concentration of

protein in a selected sample.

Ultraviolet Absorption Method

The aromatic amino acid residues (tyrosine and tryptophan) in a protein exhibit an

absorption maximum at a wavelength of 280 nm. Since the proportions of these aromatic amino

acids in proteins vary, the extinction coefficients for individual proteins also vary. However, for

the majority of proteins the extinction coefficient lies in the range 0.4–1.5; so for a complex

mixture of proteins it is a fair approximation to say that a solution with an absorbance at 280 nm

of 1.0, using a 1 cm pathlength, has a protein concentration of approximately 1 mg cm3. The

method is relatively sensitive, as it is able to measure protein concentrations as low as 10 µg cm 3,

and, unlike colorimetric methods, this technique is non-destructive, as having made the

measurement, the sample in the cuvette can be recovered and further used.
 This is useful especially when working with small amounts of protein that cannot be

allowed to go to waste. Even so, the method is subject to interference by the presence of other

compounds that absorb at 280 nm, such as nucleic acids, as they fall into this category having an

absorbance as much as 10 times that of protein at this wavelength. Hence the presence of only a

small percentage of nucleic acid can greatly influence the absorbance at this wave-length.

However, if the absorbances (A) at 280 and 260 nm wavelengths are measured, it is possible to

apply a correction factor:

Protein (mg cm-3) = 1.55 A280 - 0.76A260

An advantage of this protein assay is that it is non-destructive and can be measured

continuously, for example in chromatographic column effluents. Even greater sensitivity can be

obtained by measuring the absorbance of ultraviolet light by peptide bonds. The peptide bond

absorbs strongly in the far ultraviolet, with a maximum at about 190 nm. However, because of

the difficulties caused by the absorption by oxygen and the low output of conventional spectro-

photometers at this wavelength, measurements are usually made at 205 or 210 nm. Most proteins

have an extinction coefficient for a 1 mg cm3 solution of about 30 at 205 nm and about 20 at 210

nm. Clearly therefore measuring at these wavelengths is 20 to 30 times more sensitive than

measuring at 280 nm, and protein concentration can be measured to less than 1 µg cm-3.

However, one disadvantage of working at these lower wavelengths is that a number of

buffers and other buffer components commonly used in protein studies also absorb strongly at

this wavelength, so it is not always practical to work at this lower wavelength.

Lowry (Folin–Ciocalteau) Assay

 The Lowry Assay has been observed as one of the most commonly used methods of

protein concentration determination. The Lowry method is reasonably sensitive, detecting down

to 10 µg cm-3 of protein, and the sensitivity is moderately constant from one protein to another.

When the Folin reagent (a mixture of sodium tungstate, molybdate and phosphate), together with

a copper sulphate solution, is mixed with a protein solution, a blue-purple colour is produced

which can be quantified by its absorbance at 660 nm. The Folin reagent is reduced as the cuprous

ions are oxidized, creating Molybdenum Blue, a blue complex that absorbs light at 750 nm.

Using the Lowry method, this complex is what is used to calculate the protein concentration. The

Lowry Assay Method works on the following principle: a darker blue solution forms when the

absorbance at 750 nm increases along with the intensity of the solution. The solution’s increasing

strength is a sign that the amount of Molybdenum Blue is rising. More Molybdenum Blue in a

solution indicates more reduced Folin reagent and hence more cuprous ions in the solution. It is

deduced that cuprous ions only result when the cuprous ions react with the peptide bonds.

Therefore, the rise in cuprous ions implies that more cupric ions are being reduced as a result of

their interactions with more peptides. The more peptide linkages present, the greater the

concentration of protein.

Like the majority of colorimetric assays, care must be taken that other compounds that

interfere with the assay are not present. For this method, this includes Tris, zwitterionic buffers

such as Pipes and Hepes, and EDTA. The method is essentially based on both the Biuret

reaction, where the peptide bonds of proteins react with Cu22+ under alkaline conditions

producing Cu+, which reacts with the Folin reagent, and the Folin–Ciocalteau reaction, which

essentially involves the reduction of phosphomolybdotungstate to hetero-polymolybdenum blue

by the copper-catalysed oxidation of aromatic amino acids. The resultant strong blue colour is
therefore partly dependent on the tyrosine and tryptophan content of the protein sample. Though

the Lowry Assay Method is more sensitive than the Biuret Assay Method, it takes much longer

and is more susceptible to the effects of chemicals. Additionally, the Lowry Assay has other

disadvantages such as its pH-which is in the range of 10.0 to 10.5- as it exclusively operates

under these conditions.

References

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