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.

Comparison of protein expression patterns between hepatocellular carcinoma cell lines and a hepatoblastoma cell line

“…Silver staining has a linear dynamic range of >50-fold (26). After normalization, the coefficients of variation of the quantities of the silver-stained protein spots are between 15 and 25% (27,28), comparable to the values obtained by SYPRO Rudy staining.…”

Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Exosomal Proteins Following Epithelial-Mesenchymal Transition

“…Protein Quantitation-The protein content of exosome preparations was estimated by 1D-SDS-PAGE/SYPRO ® Ruby protein staining densitometry. This method is reproducible, has a linear quantitation range over three orders of magnitude (42), and is compatible with GeLC-MS/MS (43). Briefly, 5 l sample aliquots were solubilized in SDS sample buffer (2% (w/v) sodium dodecyl sulfate, 125 mM TrisHCl, pH 6.8, 12.5% (v/v) glycerol, 0.02% (w/v) bromphenol blue) and loaded into 1 mm, 10-well NuPAGE™ 4 -12% (w/v) Bis-Tris Precast gels (Invitrogen).…”

Protein stains for proteomic applications:Which, when, why?

“…Proteins can thus be successfully identified by peptide mass profiling using MALDI-TOF and LC-MS/MS, with even better sequence coverage than using the conventional colorimetric stains [63,64,[74][75][76][77].…”