( higher reducing potential t...)The Bio-Rad Silver Stain, Rev E

With photographic chemical-process silver stains, the silver nitrate reacts with protein sites under acidic conditions. Subsequent reduction of silver ions to metallic silver occurs by oxidation of formaldehyde under alkaline conditions. Sodium carbonate, or another base, buffers the formic acid produced by the oxidation of formaldehyde so that the silver ion reduction can continue until the protein bands appear in the gel.

With Bio-Rads silver stain, the formation of a positive image is enhanced by dichromate oxidation, which may convert protein hydroxyl and sulfhydryl groups to aldehydes and thiosulfates, thereby altering the redox potential of the protein. Complexes formed between the proteins and dichromate may also form nucleation centers for silver reduction. Basic and sulfur-containing amino acids appear to be strongly involved in the formation of complexes with silver ions.⁵³ This also appears to be true for Coomassie stains. The incorporation of these amino acids into peptide chains, as well as cooperative effects of several intramolecular functional groups, are probably required for reaction with silver ions.²⁹ Reduction of ionic to metallic silver is highly dependent on pH. This reduction step is accomplished by the alkaline organic development reagent.

Before a protein gel can be stained, the proteins must be fixed to minimize diffusion of molecules in the gel. Fixation also elutes substances from the gel that are likely to interfere with establishment of the oxidation/reduction potential differences and with silver reduction. Ampholytes, detergents, reducing agents, initiators or catalysts, and buffer ions (glycine, chloride, etc.) must be removed. Good-quality acrylamid
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