Quantitative Determination of Proteins Using Bradford Method

Angelo Rafols, James Anand Regala, Sabrina Nicolle Sarte, Ann Michelle Siao, Michael Sibulo Group 7 2C Pharmacy BioChemistry Laboratory ABSTRACT
There are several methods that can be used to determine the total concentration of protein in a sample. In this experiment we used the Bradford Assay Method. The Bradford assay is one of the most common methods used in the determination of total protein concentration of a sample. We used the Bradford reagent which is a Coomassie dye. The Coomassie dye binds to protein in acidic solution leading to an increased absorbance of the sample at 595 nm. After preparing the samples, a UV-vis spectophotometer was used to read the absorbance of the samples. A standard curve was plotted and a line equation formed. The line equation was then used to find the concentration of the given unknown.

INTRODUCTION 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 assesing protein concentrations for gel electrophoresis. Assay materials including color reagent, protein standard, and instruction booklet are available from Bio-Rad Corporation. The method described below is for a 100 l sample volume using 5 ml color reagent. It is sensitive to about 5 to 200 micrograms protein, depending on the dye quality. In assays using 5 ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 g protein. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range.[1]

Table 1. Measurement of reagents Tube No. mL BSA mL H20 1 0 1.50 2 0.10 1.40 3 0.15 1.35 4 0.20 1.30 5 0.25 1.25 6 0.30 1.20 7 0.35 1.15 8 0.40 1.10 9 0.45 1.05 10 unknown unknown Test tube 10 is the unknown sample. All these 10 samples were diluted with 1.5mL of Bradford reagent. The samples have to be mixed well and let it stand for 5 minutes.

EXPERIMENTAL
A. Compound Tested Bovine Serum Albumin(BSA) standard of 100g/mL was used as the standard. Nine(9) samples were used with different concentration of BSA and one(1) unknown. B. Procedure A series of test tubes were prepared with the following measurements: Fig 1. Samples The absorbance was read at 595 nm within an hour. The first test tube was used as the blank. An Albumin standard curve was constructed by plotting the absorbance against the concentration.

RESULTS AND DISCUSSION

The BSA used has a concentration of 100g/mL. Given the amount of BSA and amount of water, we can measure the total concentration of the protein. The following are the concentration of

the protein from the samples and their absorbance: Table 2. Concentration of protein and absorbance Tube mL BSA Conc. of Absorbance mL H20 No. protein 1 0 1.50 0 0 2 0.10 1.40 3.33 0.108 3 0.15 1.35 5 0.219 4 0.20 1.30 6.67 0.410 5 0.25 1.25 8.33 0.532 6 0.30 1.20 10 0.814 7 0.35 1.15 11.67 0.983 8 0.40 1.10 13.33 1.140 9 0.45 1.05 15 1.209 10 Unknown Unknown X 0.662 The higher the concentration of the protein, the higher the absorbance at 595 nm gathered.

Fig 2. Albumin Standard curve From the given information, we can solve for the line equation:

x-13y+0.31
From the line equation, we can solve for the concentration of protein of the unknown.

x = 13(0.662) 0.31 = 8.30

The concentration of the unknown is 8.30.

REFERENCES
[1] http://www.ruf.rice.edu/~bioslabs/method s/protein/bradford.html From Books Laboratory Manual in General Biochemistry by Crisostomo, A. et al.