Blue silver

A very sensitive colloidal Coomassie G-250 staining for proteome analysis

Giovanni Candiano, Maurizio Bruschi, Luca Musante, Laura Santucci, Gian Marco Ghiggeri, Barbara Carnemolla, Paola Orecchia, Luciano Zardi, Pier Giorgio Righetti

Research output: Contribution to journalArticle

1339 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1327-1333
Number of pages7
JournalElectrophoresis
Volume25
Issue number9
DOIs
Publication statusPublished - May 2004

Fingerprint

Proteome
Silver
Coloring Agents
Staining and Labeling
Silver Staining
Peptides
Signal-To-Noise Ratio
Bovine Serum Albumin
Electrophoresis
Aldehydes
Sodium Dodecyl Sulfate
Mass spectrometry
Limit of Detection
Polyacrylamide Gel Electrophoresis
Mass Spectrometry
Signal to noise ratio
Association reactions
Molecules
Proteins

Keywords

  • Colloidal stains
  • Human kidney tubular epithelial cells
  • Porous immobilized pH gradient gels
  • Two-dimensional maps

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Blue silver : A very sensitive colloidal Coomassie G-250 staining for proteome analysis. / Candiano, Giovanni; Bruschi, Maurizio; Musante, Luca; Santucci, Laura; Ghiggeri, Gian Marco; Carnemolla, Barbara; Orecchia, Paola; Zardi, Luciano; Righetti, Pier Giorgio.

In: Electrophoresis, Vol. 25, No. 9, 05.2004, p. 1327-1333.

Research output: Contribution to journalArticle

Candiano, G, Bruschi, M, Musante, L, Santucci, L, Ghiggeri, GM, Carnemolla, B, Orecchia, P, Zardi, L & Righetti, PG 2004, 'Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis', Electrophoresis, vol. 25, no. 9, pp. 1327-1333. https://doi.org/10.1002/elps.200305844
Candiano, Giovanni ; Bruschi, Maurizio ; Musante, Luca ; Santucci, Laura ; Ghiggeri, Gian Marco ; Carnemolla, Barbara ; Orecchia, Paola ; Zardi, Luciano ; Righetti, Pier Giorgio. / Blue silver : A very sensitive colloidal Coomassie G-250 staining for proteome analysis. In: Electrophoresis. 2004 ; Vol. 25, No. 9. pp. 1327-1333.
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AU - Candiano, Giovanni

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AU - Santucci, Laura

AU - Ghiggeri, Gian Marco

AU - Carnemolla, Barbara

AU - Orecchia, Paola

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AU - Righetti, Pier Giorgio

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AB - 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.

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KW - Two-dimensional maps

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