ESTIMATION OF PROTEINS BY BRADFORD METHOD

Aim:

To estimate the amount of protein in the given sample by Bradford Assay.

Principle

The protein in solution can be measured quantitatively by different methods. The methods described by Bradford uses a different concept-the protein‘s capacity to bind to a dye, quantitatively. The assay is based on the ability of proteins to bind to coomassie brilliant blue and form a complex whose extinction coefficient is much greater than that of free dye. 

List of Reagents and Instruments

A. Equipment

• Test tubes

• Graduated cylinder

• Weight Balance

• UV spectrophotometer

B. Reagents

• Bradford reagent: Dissolve 100mg of Coomassie-Brilliant blue G250 in 50 ml of 95% Ethanol.

• Add 100 ml of 85% phosphoric acid and make up to 600 ml with distilled water.

• Filter the solution and add 100 ml of glycerol, then make upto 1000ml.

• The solution can be used after 24 hrs.

• BSA

Procedure

• Prepare various concentration of standard protein solutions from the stock solution (say 0.2, 0.4, 0.6, 0.8 and 1.0 ml ) into series of test tubes and make up the volume to 1 ml . Pipette out 0.2ml of the sample in two other test tubes and make up the volume to 1ml.

• A tube with 1 ml of water serves as blank

• Add 5.0 ml of coomassie brilliant blue to each tube and mix by vortex or inversion.

• Wait for 10-30minutes and read each of the standards and each of the samples at 595nm.

• Plot the absorbance of the standards verses their concentration.

• Plot graph of optical density versus concentration. From graph find amount of protein in unknown sample.

Tabulation:

Sample	Volume of BSA  (ml)	Volume of Distilled water (ml)	Concentration of BSA (µg/ml)	Volume of Bradford reagent (ml)	Incubation for 10 minutes	Absorbance at 595nm
Blank	0	800	---	200
S1	10	790	5	200
S2	20	780	10	200
S3	30	770	15	200
S4	40	760	20	200
S5	50	750	25	200
Unknown	50	750	--?--	200

Notes:

     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. 

     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. 

       Assay materials including color reagent, protein standard, and instruction booklet are available from Bio-Rad Corporation. The method described below is for a 100 µl 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 µg protein. Scale down the volume for the "microassay procedure," which uses 1 ml cuvettes. Protocols, including use of microtiter plates are described in the flyer that comes with the Bio-Rad kit.