Increased Speed and Performance of Protein Staining with SYPRO® Ruby Protein Gel Stain using Hot Water Incubation

The author presents a novel rapid, low-cost staining protocol (hot water protocol) for proteins in a polyacrylamide gel using the fluorescent dye SYPRO® Ruby. Compared to the supplierrecommended protocol, the hot water protocol showed equivalent dynamic range and linearity, and a lower detection limit. Peptides prepared with in-gel digestion extracted from polyacrylamide gels stained using the hot water protocol showed a sequence coverage ratio equivalent to that obtained using the supplier-recommended protocol, and de novo sequencing could also be carried out without issue. In addition, use of the hot water protocol dramatically decreased speckling noises owing to prolonged staining time or storage of the dye solution. These results demonstrate the superiority of the hot water protocol over the supplier-recommended protocol