Detection of proteins on polyacrylamide gels using calconcarboxylic acid

We describe here a protein staining method in polyacrylamide gels with a new staining dye, 1-(2-hydroxy-4-sulfo-1-naphthylazo)-2-hydroxy-3-naphthoic acid (calconcarboxylic acid, NN). This method can be performed by both simultaneous and postelectrophoretic staining techniques. Simultaneous staining using 0.01% of NN in upper reservoir buffer eliminates the poststaining step, and thus enables detection of the proteins more rapidly and simply. In poststaining, proteins can be stained by a 30-min incubation of a polyacrylamide gel in 40% methanol/7% acetic acid solution of 0.05% NN. These techniques produced protein staining patterns identical to the ones obtained by the conventional poststaining with Coomassie blue R-250 (CB). NN staining can detect as little as 10 ng of bovine serum albumin by poststaining and 25 ng by simultaneous staining, compared to 50 ng detectable by CB poststaining. In comparing the relationship between band intensity and amount of protein, NN staining gave better linearity than CB staining.