Use of Backlit Light Plate to Enhance Visualization of Imidazole-Zinc Reverse Stained Gels

Imidazole-zinc reverse stain utilizing imidazole and zinc ions for protein visualization on electrophoresis gels was originally introduced in the 1990s (1–3). This method is based on the selective precipitation of imidazolate-zinc complex in the gel (3), except zones where proteins or other macromolecules, such as DNA (4–7) or lipopolysaccharide, are present (4,5,8,9). There are several advantages of using imidazole-zinc reverse stain in current proteomic research. First, imidazole-zinc reverse stain is highly sensitive. For protein gels, zinc reverse stain has been demonstrated to provide an equal or better staining sensitivity than Sypro Ruby stain or some silver stains (4,5). Protein, as low as 1 ng, may be detected in the gel by the reverse stain. Second, performing imidazolezinc reverse stain is exceedingly simple as compared with silver stain, which requires tedious preparation of fresh staining solutions. Imidazole-zinc reverse stain uses only two staining solutions, which can be easily diluted from stock solutions. Third, it is remarkably fast, generally taking less than 20 min to complete (4,5,10). Finally, imidazole-zinc reverse stain is fully compatible to down-stream applications, such as mass spectrometry (MS) (4,11–14), Edman sequencing (1–3), electroelution (1,2,4,6), and membrane blotting techniques (4,5). Although the dynamic range of staining for imidazole-zinc reverse stain might not be as satisfactory as for Sypro Rube stain, nowadays laboratories that are not equipped with synchronized spot pickers on their fluorescent scanners sometimes use it to restain the Sypro Ruby stained two-dimensional electrophoresis (2-DE) gels, then manually pick the interested protein spots for identification by MS. The imidazole-zinc reverse stained gel delivers an image with transparent bands, spots, and a white background. When the gel is placed above a dark Use of backlit light plate to enhance visualization of imidazole-zinc reverse stained gels