Quantitative aspects of silver deposition in proteins resolved in complex polyacrylamide gels

Deposition of silver or silver compounds in proteins separated in linear gradient polyacrylamide electrophoretic gels is influenced by protein concentration and location, polyacrylamide concentration, extent of washing after incubation in ammoniacal silver hydroxide solution, and development time in a citric acid: formaldehyde solution. Our modification of existing silver staining methods provided routine resolution of polypeptides separated in 1.0 to 1.5‐mm‐thick gels without high background staining or surface staining and with picogram detection sensitivity. Removing electrophoretic components, such as sodium dodecyl sulfate (SDS), sulfhydryl reagents (2‐mercaptoethanol, dithiothreitol and dithioerythreitol), glycerol, tris‐glycine, carrier ampholytes, urea, and acetic acid, was essential in order to attain silver staining of proteins with a negligible background. Ammoniacal silver ions or silver ions, associated and unassociated with proteins separated in polyacrylamide gels, were removed during the washing step following incubation in ammoniacal silver hydroxide solution. Loss of stain intensity at discrete protein clusters was shown to correlate with polyacrylamide concentration in gradient gels. Standardizing the washing step enhanced reproducibility of staining. Using this silver staining method and computer‐assisted image processing, it was possible to detect quantitatively as little as 27 picograms of protein per mm in 10 %‐20 % linear gradient polyacrylamide gels (1.2 mm thick). Based on the lowest detection limits for Coomassie Brilliant Blue R‐250 staining of protein (10 nanograms), this method for silver staining is at least 370‐fold more sensitive.