Pyrogallol red‐molybdate: A reversible, metal chelate stain for detection of proteins immobilized on membrane supports

Publisher Website
Google Scholar
Abstract
Certain metal complexes selectively interact with proteins immobilized on solid‐phase membrane supports to form brightly colored products. The metal chelates form protein‐dye complexes in the presence of metal ions at acidic pH but are eluted from the proteins by immersing membranes in a solution of basic pH that contains other chelating agents. The reversible nature of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting, N‐terminal and internal protein sequencing. Among the metal complexes evaluated to date, the triazine dye‐ferrous complexes (ferene S, ferrozine) and the ferrocyanide‐ferric complexes provide the most sensitive detection of proteins immobilized on membranes. While the pyrogallol redmolybdate complex is commonly used in solution‐based total protein assays, its utility as a reversible stain for proteins immobilized on membranes has not been reported. Pyrogallol red‐molybdate complexes readily stain proteins on nitrocellulose and polyvinyl difluoride membranes with similar sensitivity as ferrozine‐ferrous complexes. Analysis of charge‐fractionated carrier ampholytes and synthetic polymers of different L‐amino acids indicate that binding is prominently via protonated α and ε‐amino side chains. Carbamylation of amino groups in bovine serum albumin substantially diminishes pyrogallol red‐molybdate binding to the protein. The stain is reversible, resistant to chemical interference, and compatible with immunoblotting.