PROTEIN ASSAY USING THE BRADFORD METHOD

Jenica M. Canlas, Nikki R. Catabona, Lizbeth Aura DP. Cebrian, Aaron Dell A. Cobeng and Kristen Angela G. Cruz Group 3 2E Pharmacy Biochemistry Laboratory

ABSTRACT
A protein assay consists of two main components: the standard curve and the unknowns. In this experiment, Bradford protein assay was used to determine the actual concentration of a protein and standard curve is required. The standard used was Bovine serum albumin (BSA), and for the determination of the unknown concentration, unknown protein sample was used. Through the use of spectrophotometer, the absorbances of the unknown proteins were determined. A standard curve was drawn by plotting the 595nm (A595) versus the BSA concentration. The protein concentration of the unknown was determined by the use of linear regression analysis.

INTRODUCTION
Four spectroscopic methods are routinely used to determine the concentration of protein in a solution. These include measurement of the protein's intrinsic UV absorbance and three methods which generate a protein-dependent color change; the Lowry assay, the Smith copper/bicinchoninic assay and the Bradford dye assay. [1] In any protein assay, the ideal protein to use as a standard is a purified preparation of the protein being assayed. In the absence of such an Absolute reference protein, another protein must be selected as a relative standard. The best relative standard to use is one that gives a color yield similar to that of the protein being assayed. Selecting such a protein standard is generally done empirically. [2] Although different protein standards can be used, the group has chosen the most widely used protein as their standard Bovine Serum Albumin (BSA). The Bradford assay is a protein determination method that involves the binding of Coomassie Brilliant Blue G-250 dye to proteins (Bradford 1976). The dye exists in three forms: cationic (red), neutral (green), and anionic (blue). [2] The dye is measured at 595 nm. [3] A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The beam of light consists of a stream of photons, represented by the purple balls in the simulation shown below. When a photon encounters an analyte molecule (the analyte is the molecule being studied), there is a chance the analyte will absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of the light beam. [4] The objectives of this experiment are: (1) to determine the protein concentration of a given sample through Bradford assay, (2) to construct the albumin standard curve and (3) to determine

the concentration of unknown proteins through the use of linear regression.

EXPERIMENTAL
A. SAMPLES USED The samples used in the experiment were Bradford reagent, Bovine serum albumin (BSA) standard (100g/mL), and unknown protein sample. B. PROCEDURE In this experiment, 100g/mL concentration BSA was used as the standard protein. 9 test tubes were prepared and filled each with varied amounts of distilled water. Omitting the first test tube, the 8 test tubes were filled each with the varied amounts of the said standard. 1.5 mL Bradford reagent was added to each tube.

Figure 1: 1st to 11th test tubes The solution was mixed well by the use of vortex mixer and left to stand for 5 minutes.

Figure 2: Vortex Mixer

Each solution was placed in separate cuvettes and their absorbance was measured with use of Spectrophotometer. Albumin standard curve was constructed showing the relationship of A595 with concentration (g/mL) of each prepared standard mixtures. The total protein concentration of the sample was determined with the use of linear regression.

Table 1: The volume (mL) of the BSA stock solution, distilled water, Bradford reagent, the Concentration and the Absorbance of the 1st to the 11th test tube Through the experiment, the group was able to solve for the unknown protein concentration by using the linear regression analysis and by plotting the standard curve by absorbance against concentration. Using the standard curve, the unknown protein solution had a concentration of 500g/mL and 500g/mL. The absorbances obtained for the unknown are 0.47nm and 2.71nm. The average concentration is 500g/mL, while the average absorbance is 1.59nm.

Figure 3: Spectrophotometer and Cuvettes

RESULTS AND DISCUSSION

The Bradford protein assay is one of several simple methods commonly used to determine the total protein concentration of a sample. The method is based on the proportional binding of the dye Coomassie to proteins. Within the linear range of the assay (~5-25 mcg/mL), the more protein present, the more Coomassie binds. Furthermore, the assay is colorimetric; as the protein concentration increases, the color 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 in this assay. It is more efficient than other methods because it is faster, involves fewer mixing steps, does not require heating, and gives a more stable colorimetric response than other methods.
Test Tube # BSA D. H2O BFR Conc. (mg/mL) A 6 0.30 1.20 1.50 0.1 0.69 1 0 1.50 1.50 2 0.10 1.40 1.50 0.033 -0.000 7 0.35 1.15 1.50 0.117 0.85 0.62 8 0.40 1.10 1.50 0.133 0.73 3 0.15 1.35 1.50 0.05 0.64 9 0.45 1.05 1.50 0.15 0.88 4 0.20 1.30 1.50 0.066 0.80 10 1.5 0 1.50 0.5 0.47 5 0.25 1.25 1.50 0.083 0.68 11 1.5 0 1.50 0.5 2.71

Graph 1: Standard curve for BSA, Absorbance (nm) versus Concentration (g/mL) It shows that the absorbance has a direct relationship with the concentration of the protein sample. In other words, when the concentration is high, its absorbance is likewise high.

REFERENCES
[1] ScienceSmithEdu. Total Protein Assays http://www.science.smith.edu/departments/Bioc hem/Biochem_353/Bradford.html 12/23/12 [2] Bio-Rad. Quick Start Bradford Protein Assay http://www.biorad.com/webroot/web/pdf/lsr/liter ature/4110065A.pdf 12/23/12 [3] MSUM Biochemistry. Bradford Protein Assay http://web.mnstate.edu/provost/bradfordassay_9 6wellplate_protocol.pdf 12/23/12 [4] Spectrophotometry.html. Spectrophotometry http://www.chm.davidson.edu/vce/spectrophoto metry/Spectrophotometry.html 12/23/12