A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respectto protein detection in two-dimensional gels and identification by peptidemass profiling

Proteomic projects are often focused on the discovery of differentially exp ressed proteins between control and experimental samples. Most laboratories choose the approach of running two-dimensional (2-D) gels, analyzing them and identifying the differentially expressed proteins by in-gel digestion a nd mass spectrometry. To date, the available stains for visualizing protein s on 2-D gels have been less than ideal for these projects because of poor detection sensitivity (Coomassie blue stain) or poor peptide recovery from in-gel digests and mass spectrometry (silver stain), unless extra destainin g and washing steps are included in the protocol, in addition, the limited dynamic range of these stains has made it difficult to rigorously and relia bly determine subtle differences in protein quantities. SYPRO Ruby Protein Gel Stain is a novel, ruthenium-based fluorescent dye for the detection of proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) gels that has properties making it well suited to high-throughput pro teomics projects. The advantages of SYPRO Ruby Protein Gel Stain relative t o silver stain demonstrated in this study include a broad linear dynamic ra nge and enhanced recovery of peptides from in-gel digests for matrix assist ed laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry .