Enzyme-linked immunodetection of proteins on Coomassie blue-stained two-dimensional cellulose acetate membranes

Cellulose acetate membrane on which proteins are separated by two-dimensional electrophoresis and visualized by Coomassie staining is directly subjected to an enzyme-linked immunodetection method using a horseradish peroxidase-conjugated second antibody. The Coomassie-stained and immunochemically stained spots are distinguishable by their colors, such as blue with Coomassie blue staining and purple with horseradish peroxidase staining. Two kinds of lighting make the distinction more apparent. Coomassie-stained protein patterns are clear in the transmitted light. However, the immunochemically stained protein spots are obvious in the reflected light, in which Coomassie-stained patterns are obscure. The procedures do not require proteins to transfer onto nitrocellulose paper in contrast to proteins in polyacrylamide gel. The simple procedure excludes the nonspecific binding of the first and second antibodies to blocking proteins such as bovine serum albumin or gelatin because blocking protein is not used. With this method, matching of the Coomassie blue-stained spot with the immunochemically stained spot is done accurately and easily.