Protein Assay by the Bradford Method

Andrea Bernice M. Aguado, Bianca Manuela M. Baldonado, Emilio Luis Andres ,

Adrian C. Bayos
Group #1, 2MBio5, Department of Biological Sciences, College of Science, University of Santo Tomas, España,
Manila 1015

Abstract
Measuring the concentration of unknown proteins solution was carried out by the Bradford

protein assay which is a spectroscopic analysis in determining the concentration quantitatively.   The

experiment focuses on getting the concentration of BSA solution using Bradford method and the

spectrophotometer. Unknown solution and set of standards were prepared using different concentrations

of BSA solution, distilled water and the Bradford reagent. A standard curve was drawn by plotting BSA

concentration versus A595 of the standards. The Experimental values gathered for the 2 unknown protein

solutions were 0.12 mL for Unknown A and 0.106 mL for Unknown B. Difference in concentration was

observed from different groups based on the spectrophotometer used.

1. Introduction

Proteins are the most abundant class of biomolecules.  They are large, complex molecules that are

composed of numerous amino acids in a specific arrangement.  The Coomassie brilliant blue protein

assay, commonly known as the Bradford assay, is used because of its rapid and convenient protocol as

well as its relative sensitivity (Ernst & Zor 2010). According to Ku 2013, it was developed by Marion M.

Bradford in 1976. This method measures the presence of the basic amino acid residues which are

arginine, lysine and histidine that contributes to formation of the protein-dye complex. An assay is a

spectroscopic procedure that determines and analyzes the quantity of a protein. Without any protein in the

solution, the color will be red-brown, and when proteins bind, the pKa of the dye will shift causing the

solution to be blue. Meaning, the quantity of protein can be estimated by determining the amount of dye

in the blue ionic form. This is usually achieved by measuring the absorbance of the solution at 595 nm.

The Bradford dye will be mixed with known concentrations of a protein, Bovine Serum Albumin (BSA).

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 BSA or Bovine serum albumin is the protein used for this experiment. It is a globular protein

used for many biological applications mainly because of its stability. Moreover, it is a single polypeptide

consisting of 583 amino acids and no carbohydrates. Another reason why BSA is used is because of its

low cost and wide availability and functionality when it comes to biotechnological applications. 

Quantitative   determinations   of protein can   be done by spectroscopic methods. Ultraviolet-

Visible spectrophotometer   was used to measure the absorbance of the standard and an unknown

protein. The cuvette will contain the solution, of which the absorbance rate will be measured. The

spectrophotometer must be adjusted for each experiment because the spectrometer only produces a small

range of wavelengths at a time (Caprette, 2005b).  The Coomassie Blue absorbs 465 nm when it is not

bound to a protein. When it is bound, the Coomassie Blue absorbs 595 nm (Changes In, 1999). 

This experiment aims to determine the concentration of the Unknown protein solutions based

from the standard curve by plotting the 595 nm against the reagent blank. The percent error was must also

be calculated at the end of the experiment.

2. Experimental

2.1 Materials and Instruments used

The materials used were Bradford reagent, Unknown Protein Solution, BSA (Bovine serum

albumin, 0.2mg/mL in water), large test tubes, Serological pipettes (1 mL and 5/10 mL), Beaker,

Graduated cylinder, Test tube racks, Spectrophotometer, Vortex mixing machine and Tissue. Brands of

the product are not indicated upon doing the experiment.

2.2 Sample preparation / Tests done

The group first prepared 8 tubes wherein the set of standards and reagent blank will be placed.

Tube 1, which was the regent black consisted of 1.0 mL of distilled water. Tube 2 contained 0.8 mL of

distilled water and 0.2 mL of BSA stock solution. Tube 3 contained 0.7 mL of distilled water and 0.3 mL

of BSA stock solution. Tube 4 contained 0.6 mL of distilled water and 0.4 mL of BSA stock solution.

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Tube 5 contained 0.5 mL of distilled water and 0.5 mL of BSA stock solution. Tube 6 contained 0.4 mL

of distilled water and 0.6 mL of BSA stock solution. Tube 7 contained 0.2 mL of distilled water and 0.8

of BSA stock solution. For Tube 8, distilled water and BSA solution was not added. After preparing the

standards, the group added 5.0 mL of Bradford reagent in all of the 8 test tubes. 

For the unknown protein solution, 4 test tubes were prepared. 2 unknown proteins solution were

given in the group. The first unknown protein solution was labeled as A and B for the second. In each of

the 4 test tubes, 2 trials of unknown protein solution was prepared adding 1.0 mL in each trial and 5.0 mL

of Bradford reagent. The mixture was well mixed using a vortex mixing machine. 

The spectrophotometer was zeroed using the reagent blank. Not before 5 minutes and after 1

hour, the standards and the trials for the unknown protein solution were tested at 595 nm against the

reagent blank. Results were listed and the group drew the standard curve by plotting the concentration of

standards versus the concentration of BSA in mL.

2.3 Instrument parameters

A spectrophotometer was used in testing the standards and trials at 595 nm. The cuvette must be

wiped by tissue and rinsed by the standard being tested before and after every use.

3. Results and Discussion

The basis of this assay is that the binding of protein molecules to Coomassie Brilliant Blue G250

dye under acidic conditions results in a color change from brown to blue. The reason for the binding of

protein is the Van der Waals forces and the hydrophobic interactions. The color of the dye is proportional

to the concentration of protein wherein the solution gets bluer as concentration increases. 

In qualifying for the ideal protein standard, the quantity of the protein must be a purified sample,

however there are cases that it’s unavailable hence, is costly. BSA or Bovine serum albumin is derived

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from cow blood and is extracted using either of the three methods: cold-organic solvent fractionation,

heat shock, and ion exchange chromatography. As mentioned in the introduction, it is very useful when it

comes to different biochemical and life science applications due to its lack of effect in many chemical

reactions as it does not affect other enzymes that don’t need it for stabilization. Moreover, it gives

compatibility in the structures, standard curve linearity, and protein-protein variability that gives a

consistent result. The maximum spectrum of anionic bond held together by hydrophobic and ionic

interactions is held at 595 nm. The wavelength must be set at 595nm because it is proportional to the

amount of concentration of the protein in the sample.

The concentration of the standards was accumulated by using the formula of Beer’s Law wherein

A is the absorbance, I is the intensity of the incident light at a given wavelength, I is the transmitted
0

intensity, L the path length through the sample, and c the concentration of the absorbing species

(Cuntapay et al). The standard curve (Figure 1) was based on the concentration in the standard assay table

(Table 1) using Microsoft Excel. Based on the standard curve (Figure 1), the amount of the 2 unknown

proteins solution was solved using Linear regression to get the X seen in Figure 1.

Table 1. Standard assay table

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 Figure 1. Standard curve

A. For the x (concentration) in Unknown sample A, Trial 1 and Trial 2

Trial 1:

Given: y = 1.097

A1 = 1.097 - 0.6431/3.7946 = x

X = 0.1196

Trial 1:

Given: y = 1.079

A2 = 1.079 - 0.6431/3.7946 = x

X = 0.1148

B. For the x (concentration) in Unknown sample B, Trial 1 and Trial 2

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Trial 1:

Given: y = 1.032

B1 = 1.032 - 0.6431/3.7946 = x

X = 0.1024

Trial 2

Given: y = 1.059

B2 = 1.059 - 0.36535.8728= x

X = 0.1096

The Estimated value was calculated by getting the average mean of the 2 trials in Sample A, same

in Sample B. Estimated value for Sample A was 0.12 and 0.106 for Sample B. The percent error was

calculated using the formula:

Wherein VA represents the approximate or measured value and V E represents the exact value which was

given by the Lab professors of the class. VE for Sample A was 0.120 and 0.105 for Sample B. In order to

get the VA, the group got the average mean of each Unknown Samples and substituted it to the formula.

Percent error for Sample A was 0% and Sample B was 0.90%.

4. Conclusion and Recommendation

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 The experiment helped the group to solve the concentration of the unknown protein solution by

the use of linear regression and by plotting the standard curve. Based on the standard curve, the 2

concentration for unknown protein solutions were 0.1196 mL and 0.1148 mL for Sample A and 0.1024

mL and 0.1096 mL for Sample B. The percent error for Sample A was 0% and Sample B has 0.90%.

5. References

He, F. (2011). Bradford Protein Assay. Bio-101: e45. DOI: 10.21769/BioProtoc.45.

Ernst, O., & Zor, T. (2010). Linearization of the bradford protein assay. Journal of visualized experiments : JoVE,

(38), 1918. https://doi.org/10.3791/1918

Ku HK, Lim HM, Oh KH, Yang HJ, Jeong JS, Kim SK. Interpretation of protein quantitation using the Bradford

assay: comparison with two calculation models. Anal Biochem. 2013; 434(1):178-180. 

Caprette, David R. (May 2005b).  Principles of Spectrophotometry, from Experimental Biosciences Web site:

http://www.ruf.rice.edu/~bioslabs/methods/protein/spectrophotometer.html

Changes In Hemolymph During M. sextaDevelopment.(March 1999). Retrieved September 23, 2008, from Research

Link 2000 Web site: http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html.

Man, T. P. (n.d.). Why Is Bovine Serum the Preferred Standard for Protein Assays? Retrieved from

https://info.gbiosciences.com/blog/why-is-bovine-serum-the-preferred-standard-for-protein-assays

Cuntapay, G., Dela Cruz, K., Dela Cruz, P. K., Delloro, S. W., & Dimalanta, R. K. (n.d.). Bradford Method for

Determination of Protein Concentration. Retrieved from https://dokumen.tips/documents/49138231-

bradford-protein-concentration-assay-formal-report.html

The differences between the BCA and Bradford protein assay, which method is better? (n.d.). Retrieved from

https://www.citeqbiologics.com/difference-between-bca-bradford-protein-assays/

Phan, H. T. M., Bartelt-Hunt, S., Rodenhausen, K. B., Schubert, M., & Bartz, J. C. (2015, October 27). Investigation

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of Bovine Serum Albumin (BSA) Attachment onto Self-Assembled Monolayers (SAMs) Using

Combinatorial Quartz Crystal Microbalance with Dissipation (QCM-D) and Spectroscopic Ellipsometry

(SE). Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624694/

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