A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis

Abstract Silver staining has been the method most commonly employed for high sensitivity staining of proteins following two‐dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex‐based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.