Substituting Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis buffers improves the resolution of focusing patterns

Abstract

In a new area of postgenomics challenges, the optimization of protein identification has become a central goal in microbiochemistry. In this work, we demonstrate that the substitution of Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis (2-DE) buffers improves the focusing of whole proteins from Pseudomonas aeruginosa. This improvement of focusing concerns more particularly basic proteins. This enhancement may be attributed to a better transfer from the first to the second dimension, which probably highlights an increase in the solubility of proteins in the IPG strips. Hence, the use of an efficient tracking dye in the 2-DE buffers may enlarge protein recovery on proteome maps.

Growth and extended survival ofEscherichia coliO157:H7 in soil organic matter

“…Cells were disrupted by two cycles of freeze thaw (from -80°C to 20°C) followed by ultrasonication at 4°C (15W, 12 pulses of 3min). Cell debris was removed by centrifugation (10,000 x g, 10min), and the protein concentration was determined using the Bradford protein assay (BioRAD), with bovine serum albumin as the standard (Vilain et al 2001).…”

“…The second dimension consisted of SDS-PAGE using a 12.5% (w/v) running polyacrylamide gel and a 4.65% stacking gel (width, 18 cm; length, 20 cm; thickness, 1 mm). Gels were stained with silver (Rabilloud 1992) for spot detection and protein map construction, and with colloidal Coomassie Blue G250 for protein identification (Vilain et al 2001). Uninoculated SESOM was run on a one-dimensional SDS PAGE to check for proteins present, but following staining, none were found.…”

Synthesis and physicochemical characterization of a novel ampholytic pullulan derivative with amphiphilic behavior in alkaline media

“…The amount of 200 µg of DMAP-CMP was added to IEF buffer (final volume, 400 µl) composed of 5 M urea, 2 M thiourea, 2% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate), 2 mM tributyl phosphine, DL-1,4-dithiothreitol, 2% (v/v) carrier ampholytes (pH 3-10; Sigma), and 0.4% (w/v) Coomassie blue [24]. The first-dimension gel separation was carried out with Immobiline Dry Strips (pH 3-10, Amersham Pharmacia Biotech, Uppsala, Sweden).…”

Proteomic comparison of outer membrane protein patterns of sessile and planktonic Pseudomonas aeruginosa cells

“…Two hundred micrograms of proteins were added to IEF buffer (final volume, 400 µl) composed of 5 M urea, 2 M thiourea, 0.1% amidosulfobetaine (ASB 14), 2 mM tributyl phosphine, 2% (v/v) carrier ampholytes (pH 4-7; Sigma), 0.4% (w/v) Coomassie brilliant blue (Vilain et al, 2001). Two hundred micrograms of proteins were added to IEF buffer (final volume, 400 µl) composed of 5 M urea, 2 M thiourea, 0.1% amidosulfobetaine (ASB 14), 2 mM tributyl phosphine, 2% (v/v) carrier ampholytes (pH 4-7; Sigma), 0.4% (w/v) Coomassie brilliant blue (Vilain et al, 2001).…”