Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.
A 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.
Candiano
Giovanni
G
Laboratory on Physiopathology of Uremia, G. Gaslini Children's Hospital, Genova, Italy.
Bruschi
Maurizio
M
Musante
Luca
L
Santucci
Laura
L
Ghiggeri
Gian Marco
GM
Carnemolla
Barbara
B
Orecchia
Paola
P
Zardi
Luciano
L
Righetti
Pier Giorgio
PG
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007668:Kidney / Q000737:chemistry; D010756:Phosphoric Acids / Q000737:chemistry; D012394:Rosaniline Dyes / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000032:analysis*; D016622:Silver Staining
D010756:Phosphoric Acids; D012394:Rosaniline Dyes; D012710:Serum Albumin, Bovine; C030242:phosphoric acid; C004692:coomassie Brilliant Blue