Ultrasensitive silver‐based color staining of polypeptides in polyacrylamide gels

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Abstract
A color development system for staining polypeptides in one‐ and two‐dimensional polyacrylamide gel electrophoresis is described. The basis of the Process involves the complexing of silver with polypeptides reactive centers. The reaction is initiated by placing a polypeptide‐containing gel, previously equilibrated with an appropriate concentration of silver nitrate, into a reducing solution that contains sodium hydroxide, sodium borohydride, and formaldehyde. After an appropriate time in the reducing solution, the gel is equilibrated through two changes of an enhancing solution that contains sodium carbonate. The sodium carbonate is necessary for optimal color appear in the polypeptide ‐silver complexes after several hours in the enhancing solution and are best appreciated while viewing over a fluorescent light box that radiates light at 5000°K. The color of each polypeptide‐silver complex is clearly visible above the light background of the stained polyacrylamide gel. Colors of stained polypeptide are blue, green, yellow, and red. Subtle shades of colors also appear and thereby allow easy discrimination of overlapping spots of polypeptides in a two‐dimensional gel. To illustrate the method's relative sensitively, a two‐dimensional pattern of human fibroblast polypeptides is compared with patterns of a duplicate gel that is stained with Coomassie Blue and developed by autoradiography. The sensitivity of the silver stain process is superior to Coomassie Blue and is comparable to autoradiography after in corporation of conventional levels of 35S‐ methionine. The utility of the procedure for identifying and characterizing human proteins is illustrated by staining human proteins is illustrated by staining human plasma and platelet polypeptides after two‐dimensional gel electrophoresis. The gel electrophoresis color development system consists of steps that are simple, reproducible, and sensitive, and most importantly, which yield colored polypeptide‐silver complexes that are reproducible from gel to gel and tissue to tissue.