The study of effects of several parameters on silver staining of proteins has led to the development of a staining method which is simple and reliable, requires only few stable solutions, and can be applied to all gel types such as sodium dodecyl sulfate (SDS) containing polyacrylamide or isoelectric focusing gels. It can be used for ultrathin layers (0.1-0.2 mm) or thicker slab gels (up to 3 mm). With this method not only proteins but also polypeptides ofmolecular weights as low as 2500 are detectable with high sensitivity. Comparison of isoelectric focusing and SDS-containing gels and a simple spot test on thin gel layers show that the detection sensitivity depends not so much on the type of proteins but rather on their structure. The redox properties of the gel are important for the staining mechanism.

A new modification of silver staining of proteins in sodium dodecyl sulfate polyacrylamide gels is adapted to automated staining in PhastSystem Development Unit. The use of a reduction step, after fixation, with thiosulfate in alcoholic sodium acetate buffer results in a considerable increase in sensitivity without the need for a recycling step. The detection limit is tenfold lower than in the silver staining procedure recommended so far for PhastSystem and corresponds to 0.05-0.1 ng protein per band. Total staining time with the new procedure is 75 min.

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