Quantitation of Proteins Separated by Electrophoresis Using Coomassie Brilliant Blue

Abstract

Different electrophoretic techniques are widely used for analyzing mixtures of proteins. Most important today are sodium dodecyl sulfate (SDS) gel electrophoresis, isoelectric focusing, and two-dimensional techniques. It is often desired to determine the quantity of protein in single or multiple bands or spots. Probably the most common method for this purpose is densitometric scanning (1,2). Other approaches are based on dye elution with a wide variety of solvents, such as pyridine (3), methanol (4), NaOH (5), or alcoholic solutions of detergents (6), followed by photometric determination of dye concentration. Other techniques rely on solubilization of gels prepared with special crosslinkers (7,8). Most of these methods overcome some of the problems of densitometry, but show some limitations and drawbacks, such as the use of toxic solvents, limited stability of the dye in the solvent or limited range of protein concentration, no background correction, or limitation to special electrophoretic techniques.