DOUBLE STAINING OF COOMASSIE BLUE-STAINED POLYACRYLAMIDE GELS BY IMIDAZOLE SODIUM DODECYL-SULFATE ZINC REVERSE STAINING - SENSITIVE DETECTION OF COOMASSIE BLUE-UNDETECTED PROTEINS

The sensitivity, simplicity, and relative rapidity of Coomassie blue s taining have made this technique the method of choice for routine dete ction and quantitative analysis of gel electrophoresis-separated prote in bands in many applications. To extend the usefulness of this techni que, we have developed a new double-staining method for visualizing SD S-PAGE-separated protein bands that were undetected by Coomassie blue staining of the gel. Coomassie blue-stained gels are washed in distill ed water (15 min, two times) and then subjected to imidazole-zinc reve rse staining. As a result of the method, a homogeneous white-stained b ackground is generated and two types of protein bands can be observed: (a) typical Coomassie blue-stained bands, which appear superposed on larger transparent bands reverse-stained (transparent) bands, which we re previously undetected by the Coomassie blue staining. The method is rapid, simple, and reproducible and double-stained gels can be kept i n distilled water for months without loss of the protein pattern. The overall sensitivity is high (e.g., 1.6 ng for recombinant streptokinas e, 47 kDa) over a wide range of protein molecular weights (10 to 100 k Da) and independent of the degree of Coomassie blue destaining of the gel. Furthermore, a mechanism offering a consistent explanation for th e role of imidazole, SDS, and zinc in the reverse staining of gels, pa rticularly after Coomassie blue staining is proposed. (C) 1995 Academi c Press, Inc.