Double Staining of Coomassie Blue-Stained Polyacrylamide Gels by Imidazole-Sodium Dodecyl Sulfate-Zinc Reverse Staining: Sensitive Detection of Coomassie Blue-Undetected Proteins

✍ Scribed by C. Fernandezpatron; E. Hardy; A. Sosa; J. Seoane; L. Castellanos

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 621 KB
• Volume: 224
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1039

✦ Synopsis

The sensitivity, simplicity, and relative rapidity of Coomassie blue staining have made this technique the method of choice for routine detection and quantitative analysis of gel electrophoresis-separated protein bands in many applications. To extend the usefulness of this technique, we have developed a new double-staining method for visualizing SDS-PAGE-separated protein bands that were undetected by Coomassie blue staining of the gel. Coomassie blue-stained gels are washed in distilled water ( (15 \mathrm{~min}), two times) and then subjected to imidazole-zinc reverse staining. As a result of the method, a homogeneous white-stained background is generated and two types of protein bands can be observed: (a) typical Coomassie blue-stained bands, which appear superposed on larger transparent bands; and (b) reverse-stained (transparent) bands, which were previously undetected by the Coomassie blue staining. The method is rapid, simple, and reproducible and doublestained gels can be kept in distilled water for months without loss of the protein pattern. The overall sensitivity is high (e.g., (1.6 \mathrm{ng}) for recombinant streptokinase, (47 \mathrm{kDa}) ) over a wide range of protein molecular weights ( 10 to (100 \mathrm{kDa}) ) and independent of the degree of Coomassie blue destaining of the gel. Furthermore, a mechanism offering a consistent explanation for the role of imidazole, SDS, and zinc in the reverse staining of gels, particularly after Coomassie blue staining is proposed. ċ 1995 Academic Press, Inc.