Protein Assay Using Bradford Method

PROTEIN ASSAY USING BRADFORD METHOD Josette Kristelle C. Allas, Kristine D. Añonuevo, Marc Eldrich T. Barroa, John Laurence A. Bautista, Ronel Mark B. Bautista Group 1 2A Medical Technology Biochemistry Laboratory

ABSTRACT

This experiment is an attempt to exemplify the usage of the Bradford Assay technique by determining the concentration of a given protein, this method implies the use of the acidic Coomassie dye as a colouring agent (the solution is red-brown in it¶s acidic solution. When protein binds, the pKa of the dye shifts causing the dye to become blue) and BSA to prepare standards. A standard is the µcontrol¶ of this experiment, our standard in this case was created by BSA. Since it is clear that concentration is directly proportional to absorption, UV-Vis Spectrophotometer was used to measure the absorption of the analytes. We already know the protein concentration of the analytes; therefore by preparing samples with known amounts of protein and comparing their absorbances with that of the unknown, the unknown quantity can be estimated. In a graphical representation of the relationship of absorption and concentration, a Standard Calibration Curve was developed and then used to infer the unknown concentration of the sample.

I. INTRODUCTION The Bradford protein assay was introduced to us as one of the methods to determine protein concentration in the mixture. The method is based on the proportional binding of the dye Coomassie to proteins, the more protein present, the more Coomassie binds and it produces a significant change in colour of the mixture. And since the assay is colorimetric, we learned that as the protein concentration increases, the colour of  the test sample becomes darker. Coomassie absorbs at 595 nm. The protein concentration of  a test sample is determined by comparison to that of a series of protein standards known to reproducibly exhibit a linear absorbance profile when plotted in a graph. Although different protein standards can be used, we have chosen the most widely used protein as our standard Bovine Serum Albumin (BSA).

solution. Protein assays are quantitative if the protein to be assayed is available in sufficient quantity such that one is able to use it to create a standard curve. If this can not be achieved, then a standard protein, such as albumin, may be used for a standard curve with the understanding that the results on the unknown protein are semiquantative. 5Because most proteins are not available in large quantities, standard curves for protein assays are typically based on the use of  either bovine serum albumin (BSA) or bovine gamma globulin (IgG).

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The Bradford assay is faster, involves fewer mixing steps, does not require heating, this assay has been found to be useful for peptides and proteins having molecular weights greater than approximately 3,000-5,000, depending on the presence of charged groups. Interfering substances include many detergents and basic buffers; there are modifications of this assay that reduce or eliminate the effects of many interfering substances. 2

Protein assays are designed to measure the total protein in a solution; that is all of the proteins in

Structural formula of Coomassie dye variant (most likely) used in our Bradford Method Coomassie dye. Dr. Marion Bradford was the first scientist to ever use Coomassie dye to determine protein concentration in his solution. 1 In the acidic environment of the reagent, protein binds to the coomassie dye.

Advantages of using Coomassie dye in Bradford Assay: 5Coomassie dye binding assays are the fastest and easiest to perform of all protein assays. 2The assay is performed at room temperature and no specials equipment is required. Standard and unknown samples are added to tubes containing preformulated Coomassie assay reagent and the resultant blue color is measured at 595 nm following short room temperature incubation. Disadvantages include 3 incompatibility with surfactants at concentrations routinely used to solubilize membrane proteins. 4The presence of a surfactant in the sample, even at low concentrations, causes precipitation of the reagent. In addition, the Coomassie dye reagent is highly acidic, so proteins with poor acidsolubility cannot be assayed with this reagent.

II. EXPERIMENTAL After procuring our own Bradford reagent and BSA aliquot onto our work table, we prepared 9 test tubes and filled each with varied amounts of  distilled water. The first test tubes had slightly more than the latter ones, test tubes 2 and 3 for instance, had a difference of approximately 0.5ml, and the rest followed in similar fashion. We poured BSA on 8 test tubes (omitting the first) again in varied amounts, this time the preceding test tubes had more BSA than the latter ones, effectively making them less concentrated with BSA. Each test tubes was mixed thoroughly, 1.5ml of Bradford reagent was then added into all test tubes. The test tubes stood undisrupted for 5 minutes then we transferred their contents into 9 cuvettes, one additional cuvette contained the sample. The prepared cuvettes were taken to Laboratory 17 downstairs and we measured their absorbance using UV-Vis Spectrophotometer An albumin standard curve was made showing the relationship of A545 with concentration of each prepared standard mixtures. Points were connected in linear regression, in doing so, our group determined the total protein concentration of the sample.

III. RESULTS AND DISCUSSIONS

TEST TUBE NO.

ABSORBANCE

1

0

2

0.842

3

0.959

4

1.039

5

1.073

6

1.080

7

1.130

8

1.138

9

1.087

The values of the absorbance of the protein standards steadily increases as the concentration of the protein standards increases, thus, their points were located close to the best fit line. Therefore, it can be seen that there is a direct relationship between the absorbance of a protein sample and its protein concentration.

Standard Bovine Serum Albumin Curve 2 1.5

y = 0.1112x R² = 0.3704

1 0.5 A595

0

5 10 y = 0.070x0+ 0.395 R² = 0.674 Concentration BSA, µg/mL

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The Coomassie Blue is a brown liquid that turns blue when attached to a protein; the more intense the blue color, the more protein. By setting the Spectrophotometer to 595 nm of  wave length, we effectively measured how much light has been absorbed by the sample.

IV. REFERENCES 1

Krohn, R.I. (2001). The Colorimetric Determination of Total Protein, Current Protocols in Food Analytical Chemistry , B1.1.1-B1.1.27, John Wiley & Sons, Inc. 2

Hopkins, (1993) Maton et. Al. Human Biology and Health. Englewood Cliffs, Michigan, USA: Prentice Hall 3

Kruger,N.(2003) The Protein Protocols Handbook B.14-16, 4

www.piercenet.com/browse.cfm?fldID=876562B 0-5056-8A76-4E0C-B764EAB3A339 Retrieved January 10 2011 5

http://www.tutorvista.com/content/chemi stry/chemistry-ii/Bradford.htm Retrieved 1/10/2011