Sensitive silver staining of protein in sodium dodecyl sulfate-polyacrylamide gels using an azo dye, calconcarboxylic acid, as a silver-ion sensitizer

A highly sensitive silver staining method for detecting proteins in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was developed. It is based on the silver nitrate staining method but also employs an azo dye, calconcarboxylic acid (NN), as a silver‐ion sensitizer. It increases silver binding on protein bands or spots by the formation of a silver‐dye complex and also increases the reducing power of silver ions to metallic silver by NN itself with formaldehyde. After a 2 h gel fixing step, the protocol including sensitization, silver‐ion impregnation, and reduction steps can be completed in 1 h. The sensitivity is superior to that of silver stain with glutardialdehyde as a silver‐ion sensitizer. The detection limit of NN‐silver stain is 0.05–0.2 ng protein. Considering the high sensitivity without using glutardialdehyde, the NN‐silver stain would be useful for routine silver staining of proteins.