Protein Estimation

Quantitation of the amount of protein in liver extracts was determined using the calorimetric method of Bradford (1976). The Bradford method depends on quantitating the binding of a dye, Coomassie Brilliant Blue, to an unknown protein and comparing this binding to that of different amounts of a standard protein, usually bovine serum albumin (BSA). Ref.: Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248-254. MATERIALS AND SOLUTIONS 1. 1 mg/ml Bovine Serum Albumin (BSA) 2. 0.15 M NaCl 3. Coomassie Brilliant Blue Solution (200 ml) composed of: - Coomassie Brilliant Blue G-250, 20 mg - 95% Ethanol, 10 ml - 85% Phosphoric acid, 20 ml - Deionized H2O, 170 ml The solution is mixed thoroughly, then filtered through Whatman No.1 filter paper and Stored at 4oC.

PROCEDURE The following components were mixed into microfuge tubes (in duplicates). Tube No. 1 2 3 4 5 6 (diluted extract) BSA (1 mg/ml) 0 l (0 g) 5 l (5 g) 10 l (10 g) 15 l (15 g ) 20 l (20 g) 25 l (25 g) 5 l 0.15 M NaCl 100 l 95 l 90 l 85 l 80 l 75 l 95 l Coomassie Brilliant Blue Solution 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml

BSA concentration (g/ml) 0 5 10 15 20 25

595 nm Absorbance 0.000 0.297 0.566 0.817 0.991 1.230

The mixture was allowed to stand 5 minutes at room temperature. Then the absorbance at 595 nm was determined and used to plot a best-fit straight line standard curve by plotting absorbance at 595 nm versus protein concentration.

To determine the protein concentration of liver samples from their absorbance, the standard curve was used to find the concentration of standard that would have the same absorbance as the sample using the straight line equation.

Y= 0.0485x + 0.0442
1.4

Absorbance at 595 nm

1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 5 10 15 20 25 30

BSA (g/mg)