Refinement of the Coomassie blue method of protein quantitation: A simple and linear spectrophotometric assay for ≤0.5 to 50 μg of protein

Abstract

The Coomassie brilliant blue G assay for proteins described by Bradford (1976) ( Anal. Biochem. 72, 248) was reexamined. It was found that the extinction coefficient of the dye-protein complex solution remained constant over the protein concentration range of 0.8 to 10 μg/ml of solution. This unchanging extinction coefficient, A 595 = 0.60 ± 0.01 10 μ g of protein/ml of solution, enhances both the sensitivity and versatility of the assay. Selection of a volume of dye-reagent (0.5 to 5.0 ml) which dilutes the protein sample to a final concentration of 0.8 to 10 μg/ml permits the application of Beer's Law for accurate determinations of ≤0.5 to 50 μg of protein. A combination of Bradford's study and the present one indicates that most common laboratory reagents and chemicals exert little or no influence on the A 595 of the dye-reagent.