Towards stoichiometric silver staining of proteins resolved in complex two‐dimensional electrophoresis gels: Real‐time analysis of pattern development

Toward the ultimate goal of deriving quantitative protein data from silver‐stained proteins resolved in complex two‐dimensional electrophoresis gels we have performed computerized real‐time analysis during pattern protein “silver stain” development. Three points emerged from this study: (i) Development time is a far more important variable than originally envisioned. (ii) Previous studies which, on the basis of a single development time, sought to relate silver stain slope and threshold properties to amino acid composition are non‐informative since it will be shown that slope values change markedly with development time, (iii) The combination of real‐time analysis and total protein load consideration allows, for the first time, protein “spots” to be grouped into categories, which in turn aid in quantification.