Blue silver: A very sensitive colloidal Coomassie G‐250 staining for proteome analysis

Electrophoresis - Tập 25 Số 9 - Trang 1327-1333 - 2004
Giovanni Candiano1, Maurizio Bruschi1, Luca Musante1, Laura Santucci1, Gian Marco Ghiggeri1, Barbara Carnemolla2, Paola Orecchia2, Luciano Zardi3, Pier Giorgio Righetti4
1Laboratory on Physiopathology of Uremia, G. Gaslini Children’s Hospital, Genova, Italy
2National Cancer Research Institute, Genova, Italy
3Institute G. Gaslini Chldren's Hospital, Genova, Italy
4University of Verona, Department of Agricultural and Industrial Biotechnologies, Verona, Italy

Tóm tắt

AbstractA modified Neuhoff's colloidal Coomassie Blue G‐250 stain is reported, dubbed “blue silver” on account of its considerably higher sensitivity, approaching the one of conventional silver staining. The main modifications, as compared to Neuhoff's protocol, were: a 20% increment in dye concentration (from 0.1% up to 0.12%) and a much higher level of phosphoric acid in the recipe (from 2% up to 10%). The “blue silver” exhibits a much faster dye uptake (80% during the first hour of coloration, vs. none with a commercial preparation from Sigma). Even at equilibrium (24 h staining), the “blue silver” exhibits a much higher sensitivity than all other recipes, approaching (but lower than) the one of the classical silver stain. Measurements of stain sensitivity after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of bovine serum albumin (BSA) gave a detection limit (signal‐to‐noise ratio > 3) of 1 ng in a single zone. The somewhat lower sensitivity of “blue silver” as compared to classical silvering protocols in the presence of aldehydes is amply compensated for by its full compatibility with mass spectrometry of eluted polypeptide chains, after a two‐dimensional map analysis, thus confirming that no dye is covalently bound (or permanently modifies) to any residue in the proteinaceous material. It is believed that the higher level of phosphoric acid in the recipe, thus its lower final pH, helps in protonating the last dissociated residues of Asp and Glu in the polypeptide coils, thus greatly favoring ionic anchoring of dye molecules to the protein moiety. Such a binding, though, must be followed by considerable hydrophobic association with the aromatic and hydrophobic residues along the polypeptide backbone.

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Electrophoresis

Tập 25 Số 9

1327-1333

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