PROTEIN REVERSE STAINING - HIGH-EFFICIENCY MICROANALYSIS OF UNMODIFIED PROTEINS DETECTED ON ELECTROPHORESIS GELS

A methodology is presented for efficiently gaining structural informat ion from electrophoresed proteins after on-gel detection by imidazole- sodium dodecyl sulfate-zinc reverse staining. As a consequence of reve rse staining, (a) protein bands arise transparent against a deep white -stained background, limits of detection being in the femtomole range; (b) there is no loss of image when the gel is kept in distilled water (even during years); and (c) protein bands result immobilized, i.e., they do not diffuse upon gel storage. To recover reverse-stained prote ins or fragments thereof from the gel, the immobilization of bands mus t first be abrogated by chelating the zinc ions from stain (protein mo bilization). We had originally described mobilization at low pH by usi ng citric acid. Here, we improve this procedure regarding the protein electrotransfer. We demonstrate that mobilization is efficiently done at neutral to alkaline pH by short-term (5 to 10 min) incubation of th e gel in a buffer containing glycine or dithiothreitol prior to transf er. Moreover, mobilization was most simply performed by just adding th e zinc chelating agent to the transfer buffer. Reverse staining and th e new mobilization procedure made electrotransferring single protein b ands from gel onto small-sized (13 X 5 mm(2)) PVDF membrane pieces in mini sandwich-like assemblies practical. Equipment is described for th e protein electroblotting in such minisandwiches. Microsequence analys is of the electroblotted proteins showed initial yields in the range o f those achieved when the transfer was done from unstained control gel s. Protein bands kept in the reverse-stained gel for prolonged time pe riods (even for as long as 2 years) could be similarly analyzed. Rever se-stained proteins were amenable to further analyses, e.g., tryptic d igestion, cysteine reduction and carboxymethylation, or N-terminal deb locking. Current results demonstrate that, after mobilization, protein s from reverse-stained gels can be manipulated for analysis as they we re in unstained gels. The reverse-staining technique could now be inco rporated into the current laboratory practice without modification of the standard methods for electrophoresis, electroblotting, and protein microanalysis. (C) 1995 Academic Press, Inc.