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PROTEIN ASSAY USING THE BRADFORD METHOD

Jiro A. Laxamana, John Patrick T. Lizarondo, Naim G. Macasalong, Don Gilson L. Maderaso, Xyza S. Malenab Group 4 2A-Medical Technology Bio Chemistry

ABSTRACT
There were three proteins that were isolated. Group 4 was assigned to isolate gluten from wheat flour by difference in solubility. After the isolation of the protein the group was asked to know the concentration of the protein. In order to quantitatively know the concentration of the protein the group used the Bradford method. The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. The Bradford protein assay was developed by Marion M. Bradford. Albumin standard curve was constructed and the unknown concentration of the protein was determined using linear regression analysis. The graph studied showed the direct relation of Bovine Serum Albumin concentration to its absorbance.

INTRODUCTION
The Bradford method assay is commonly used to determine the total protein concentration of a sample. The method is based on the binding of Coomassie dye to proteins in acidic solution leading to an increased absorbance of the sample at 595 nm. The assay is sensitive to about 20 to 200 g protein. The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions and assessing protein concentrations for gel electrophoresis. The assay is useful since the extinction coefficient of a dyealbumin complex solution is constant over a 10fold concentration range. The Bradford protein assay is one of several simple methods commonly used to determine the total protein concentration of a sample. Furthermore, the assay is colorimetric; as the protein concentration increases, the color of the test sample becomes darker. 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. Although different protein standards can be used, we have chosen the most widely used protein as our standard - Bovine Serum Albumin (BSA).

B. Procedure 1. Preparation of the solution Prepare a series of test tubes as follows mL 0 0.10 0.15 0.20 0.25 standard mL H20 1.50 1.40 1.35 1.30 1.25

0.30 1.20

0.35 1.15

0.40 1.10

0.45 1.05

2. Adding of Bradford reagent With each test tube add 1.5 mL Bradford reagent. Mix well. Let it stand for 5 minutes 3. Preparation of cuvette Prepare cuvettes for spectrophotometry. Each cuvette was cleaned using distilled water and using clean tissue paper to wipe it. 4. Reading the absorbance Use one cuvette as a blank then place it to a spectrophotometer to be able to read its absorbance. Each cuvette must be used in the spectrophotometer to be able to use the data for the group to be able to plot and determine the concentration of the protein. 5. Construction of albumin standard curve Construct the albumin standard curve by plotting against concentration and determine the concentration of proteins in the protein sample. Linear regression may also be used to determine the concentration.

EXPERIMENTAL
A. Compounds Tested Bradford reagent Bovine serum albumin standard Gluten

RESULTS AND DISCUSSION


The Bradford assay is commonly used to determine the total protein concentration of as ample. The method is based on the binding of Coomassie dye to proteins. The acidic environment of the reagent results in a spectral shift from the reddish brown of the dye, which has a maximum absorbance of 465 nm, to the blue form of the dye, which has a maximum absorbance of 610 nm. The difference between the two forms of the dye is greatest at 595 nm. In determining the total protein concentration of the sample, the dilution formula was used to compute for the total protein concentration. Test tube no. 1 2 3 4 5 6 7 8 Concentration 0 3.3 5 6.7 8.3 10 12 13 absorbance 0 0.842 0.959 1.039 1.073 1.08 1.13 1.138 Calculations A595 of sx = 1.087

= 9.9 g/mL

9.9 g/mL x

= 66 g/mL

Figure 1: concentration and absorbance using spectrophotometer

REFERENCE
Laboratory Manual in General Biochemistry Quezon City: C & E Publishing, Inc. Milio, f.R., & Loffredo, W.M. (1995). Qualitative Testinf for amino acids. Retrieved from http://jan.ucc.nau.edu/~jkn/ 235l7TLC%20Analgesics.htm Ganong, B. (2008). Biochemistry: course description. Retrieved from http://Faculty .mansfield.edu/bganong/341.html