Protein Assay by the Bradford Method

-------------BIOCHEMISTRY --------------

Biochemistry Laboratory – CH600 (2008-2009) Experiment 3

Protein Assay by the Bradford Method *Michelle Dy Sim, Gellina Ann Ram Suderio, Jonnah Kristina Chua Teope Department of Biology, 3Biology-6, College of Science University of Santo Tomas, España Street, Manila 1008 December 12, 2008 Abstract: The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. This experiment aims to determine the concentration of the unknown protein solution and to draw the standard curve by plotting the 595nm (A595) against a reagent blank. Standards were prepared by adding 0.3 and 0.4mL of BSA stock solution. Distilled water was added to each of the test tube to bring the volume to 1mL. For the determination of the unknown concentration, 1mL of the unknown protein sample was used. Through the use of the spectrophotometer, the absorbances (in nm) for the unknown proteins were determined. A standard curve was drawn by plotting the A595 versus the BSA concentration. The concentrations of the unknown proteins were solved by using linear regression. The result obtained for the concentration of the unknown for trials 1 and 2 are 106.117µg/mL and 88.335µg/mL. The average concentration is 97.226µg/mL. The average absorbance is 0.2929nm. Keywords: • Protein • Bradford Assay Method • Spectrophotometer • BSA standard (bovine serum albumin)

I. Introduction There is no single protein assay method that yields absolutely accurate results. The only true and accurate method for determining protein concentration is by acid hydrolyzing a portion of the sample and then carries out amino acid analysis on the hydrolyzate. But, this method is time-consuming. Each method and assay has its own disadvantage and limitations. The most commonly used assays are the Ultraviolet Absorbance, Lowry Assay, BCA assay and the Bradford Assay. The UV absorbance monitors the absorbance of aromatic amino acids, tyrosine and tryptophan or if the wavelength is lowered, the absorbance of the peptide bond. It is quick, with the

samples that can be recovered. But, it is also highly susceptible to contamination by buffers, biological materials and salts. The Lowry Assay or enhanced copper since it reduces Cu+2 to Cu+1 , sensitive over a wide range and is the most commonly referenced procedure for protein determination but, it also takes a considerable amount of time. And the BCA assay or the bicinchoninic acid which is less susceptible to interference from common buffer substance and is very sensitive and rapid if you use elevated temperatures, but, the reaction does not go to completion when performed at room temperature and dilution is often necessary for concentrated protein samples. The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. It is dependent on the amino acid composition of the measured protein. It is more efficient than other methods because assay it is faster, involves fewer mixing steps, does not require heating, and gives a more stable colorimetric response than other methods. The Bradford assay is faster, involves fewer mixing steps, does not require heating, and gives a more stable colorimetric response. Its response is prone to influence from non protein sources and becomes progressively more nonlinear at the high end of its useful protein concentration range. The response is also protein dependent, and varies with the composition of the protein. These limitations make protein standard solutions necessary. A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam through a sample and measuring the intensity of light reaching a detector. The beam of light consists of a stream of photons. When, a photon encounters an analyte molecule, there is a chance the analyte will absorb the photon. This absorption reduces the number of photon in the beam of light, thereby reducing the intensity of the light beam. The objective of this experiment is to determine the concentration of the unknown protein solution and to draw the standard curve by plotting the 595 nm (A595) against a reagent blank.

II. Methodology The class was divided into two teams. Team A comprised of groups 1 to 5. Team B comprised of groups 6 to 9. The members of this group were assigned to perform only test tube numbers 3 and 4. Standards were prepared. For test tubes number three and four, 0.3mL and 0.4mL of the BSA stock solution were used. Then, distilled water was added to each tube to bring the volume to 1mL. To each of these test tubes, 5mL of Bradford reagent was added. Table 1. The volume (mL) of each test tube, BSA stock Solution (mL), distilled water (mL), Bradford reagent(mL)

Test Tube # BSA Stock, mL Distilled Water, mL Bradford Reagent, mL

3 0.3 0.7 5

4 0.4 0.6 5

For the unknown protein solution, 1mL was used. Then, 5mL of the Bradford reagent was added. The spectrophotometer was then zeroed using a test tube 1 as the reagent blank. After five minutes, but before one hour, the absorbance of the standards and the unknown protein solution were read at 595 nm (A595). A standard curve was drawn by plotting the A595 versus the concentration of BSA.

III. Results and Discussion A. Results Table 2. The volume (mL) of the BSA stock solution, distilled water, Bradford reagent, the Concentration and the Absorbance of the 1st to the 6th test tube

Test Tube # BSA Stock, mL Distilled Water ,mL Bradford Reagent, mL Concentration µg/mL Absorbance

1 0 1 5 0 -0.0002

2 0.2 0.8 5 40 0.1404

3 0.3 0.7 5 60 0.2099

4 0.4 0.6 5 80 0.2657

5 0.5 0.5 5 100 0.3117

6 0.6 0.4 5 120 0.3632

Table 3. The volume (mL) of the BSA stock solution, distilled water, Bradford reagent, the Concentration and the Absorbance of the 7th, 8th and the 2 tubes containing the unknown

Test Tube # BSA Stock, mL Distilled Water ,mL Bradford Reagent, mL Concentration µg/mL Absorbance

7 0.8 0.2 5 160 0.4624

COMPUTATIONS: A = 0.06923 B = 0.0023 y = A + Bx y = 0.06923 + 0.0023x Concentration of Unknown Trial # 1: Substitute (y = 0.3133) 0.3133 = 0.06923 + 0.0023x X = 0.3133 – 0.06923 0.0023 X = 106.117 µg/mL Concentration of Unknown Trial # 2: Substitute (y = 0.2724) 0.2724 = 0.06923 + 0.0023x X = 0.2724 – 0.06923 0.0023 X = 88.335 µg/mL Average Concentration: Average concentration = 106.117 + 88.335 2 = 97.226 µg/mL Average Absorbance: Average Absorbance = 0.3133 + 0.2724 2 = 0.2929nm

8 1 0 5 200 0.5129

Unknown Trial # 1 5 106.117 0.3133

Unknown Trial # 2 5 88.335 0.2724

The Standard Curve of A 595 Versus The Concentration of BSA 0.6

200, 0.5129

0.5

160, 0.4624

0.4

absorbance (y)

120, 0.3632 100, 0.3117 97.226, 0.2929

0.3

80, 0.2657

60, 0.2099

0.2

40, 0.1404 0.1

0

0, -0.0002 0

50

100

150

200

250

-0.1 concentration (x)

Graph 1. Standard curve for BSA, Absorbance (nm) versus Concentration (µg/mL)

B. Discussion The Bradford assay, a colorimetric protein assay, is based on an absorbance shift in the dye Coomassie when the previously red form Coomassie reagent changes and stabilizes into Coomassie blue by the binding of protein. During the formation of this complex, two types of bond interaction take place: the red form of coomassie dye first donates its free proton to the ionizable groups on the protein, which causes a disruption of the protein’s native state, consequently exposing its hydrophobic pockets. These pockets on the protein’s tertiary structure bind non-covalently to the non-polar region of the dye via Van der Waals forces, positioning the positive amine group in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. Binding of the protein stabilizes the blue form of coomassie dye, thus the amount of complex present in solution is a measure for the protein concentration by use of an absorbance reading.

Figure 1. Coomassie Brilliant Blue G-250 structure

Protein Red (470 nm) H+

Green (650 nm) H+

Blue (590 nm)

Blue-Protein (590 nm)

Figure 2. Equilibrium of Coomassie brilliant blue G-250

The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range.

Because the Bradford assay essentially measures the amount of arginine and hydrophobic amino acid residues, the amino acid composition can alter the concentration-absorbance curve depending on the percentage of arginine or hydrophobic amino acids in each protein. It is therefore necessary to use a standard (e.g. BSA-- Bovine Serum Albumin) whose protein closely matches the measured protein in composition The dye reagent reacts primarily with arginine residues and less so with histidine, lysine, tyrosine, tryptophan, and phenylalanine residues. Obviously, the assay is less accurate for basic or acidic proteins. The Bradford assay is rather sensitive to bovine serum albumin, more so than "average" proteins, by about a factor of two. Immunoglogin G (IgG - gamma globulin) is the preferred protein standard. The addition of 1 M NaOH was suggested by Stoscheck (1990) to allow the solubilization of membrane proteins and reduce the protein-to-protein variation in color yield.

Error in the experiment can be a cause of a lot of factor: the prediluted standards are conveniently packaged eliminating wasteful and sharp ampoules, and ensuring protein stability over the shelf life. Pipetting of the reagents and the dye can cause problems such as the inadequate or too much addition of both. Spectrophotometer should be down to the zero point by the reagent blank since it can be a very big factor for error in the experiment. It is also suggested that at least two reagents blank should be performed or one can use a water buffer instead. But, if the spectrophotometer was not zeroed with the blank, take the average of the blank value from the standard and unknown sample values instead. When the absorbance of protein standard and sample is very low, the 1x dye reagent may be cold from 40 storage, and then warm the dye reagent to ambient temperature. If the absorbance of the standard is acceptable, but if the absorbances of samples are very low, then the samples may contain a substance that interferes with the reaction, such as detergents or basic solutions.

IV. Conclusion

Through the experiment, the group was able to solve for the concentration of the unknown protein solution by using the linear regression method and by plotting the standard curve by absorbance versus concentration. Using the standard curve, The unknown protein solution had a concentration of 106.117µg/mL and 88.335µg/mL. The absorbance obtained for the unknown are 0.3133nm and 0.2724nm. The average concentration is 97.226µg/mL. The average absorbance is 0.2929nm. The Bradford method shows that the absorbance has a direct relationship with the concentration of the protein sample, meaning if the absorbance is high, the concentration of the sample is also high.

V. References Books: [1] Bollag, D., Rozycki M. Edelstein. Protein Methods Second Edition. New York: Wiley – Liss, 1996. [2] Boyer, Rodney. Modern Experimental Biochemistry Third Edition. California: Addison Wesley Longman Inc., 2000. [3] Davis, W. and Peck Stanley. Chemistry Eighth Edition. United States of America: Thomson Brooks Cole, 2007. [4] McMurry, John. Organic Chemistry: A Biological Approach. United States of America: Thomson Brooks Cole, 2007. [5] Walker, John M. Basic Protein and Peptide Protocol Volume 32. New Jersey: Human Press Inc., 1994. Articles: [1] Bradley J.S.C. Olson and John Markwell. Current Protocols in Protein Science. 3.4.1-3.4.29. John Wiley & Sons, Inc., 2007. [2] BRADFORD ASSAY: Generating Standard Curve – Retrieved December 9, 2008 http://molecool.wustl.edu/krolllab/Kroll_Lab_Protocols/Protein%20methods/Protein%20Assay %20Guides/Bradford%20assay-Biorad.pdf Internet Sources: [1] Bradford Protein Assay – Retrieved December 8, 2008 http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html [2] Bradford Protein Assay – Retrieved December 7, 2008 http://www.science.smith.edu/departments/Biochem/Biochem_353/Bradford.html#theory

[3] Spectrophotometry – Retrieved December 8, 2008 http://www.spectrophotometer.com/FAQ.htm