Improved protein solubility in two‐dimensional electrophoresis using tributyl phosphine as reducing agent

Abstract

In this study, dithiothreitol was replaced by tributyl phosphine as the reducing agent in both the sample solution for the first-dimensional isoelectric focusing and during the immobilised pH gradient (IPG) equilibration procedure. Tributyl phosphine improves protein solubility during isoelectric focusing, which results in shorter run times and increased resolution. Tributyl phosphine is nonionic and thus does not migrate in the IPG, therefore maintaining reducing conditions during the course of the first-dimensional separation. The increased solubility provided by the maintenance of reducing conditions gives improved focusing and decreased horizontal streaking on the subsequent second-dimension gel. The use of tributyl phosphine in the equilibration step allows the procedure to be simplified, incorporating reduction and alkylation in a single step. This is possible because, in direct contrast to dithiothreitol (DTT), tributyl phosphine does not contain a free thiol and therefore does not react with thiol-specific alkylating reagents.

Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle, Balanus amphitrite

“…Thiourea might have enhanced the solublization of the membranes or highly hydrophobic proteins in our samples, especially after mild sonication [22]. The solublization of membrane proteins is still the most challenging step in 2-DE-based proteomics approaches [23][24][25]. Nevertheless, the optimized protocol in this study resulted in a uniform spot distribution on a single protein map and we observed excellent reproducibility (Fig.…”

Proteomic Analysis of the Systemic Immune Response of Drosophila

“…A mixture composed of 0.5% Triton X-100 and 4% CHAPS was found to be the most efficient for solubilization of proteins. In order to improve the solubility of molecules, urea was replaced by a mixture of thiourea/urea (2 M/7 M), reported to have a much higher power of solubility (23), and DTT was replaced by tributylphosphine (5 mM), a more powerful reducing agent (24). However, these reagent modifications had no obvious benefit for the 2D maps.…”

Proteomics of the Chloroplast: Systematic Identification and Targeting Analysis of Lumenal and Peripheral Thylakoid Proteins

“…The focused strips were equilibrated in a solution containing 6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 6.8, 5 mM TBP, and 2% SDS (w/v) for 20 min (Rabilloud et al, 1997;Herbert et al, 1998). Separation in the second dimension was conducted at room temperature on gradient Tricine-SDS gels (8 to 16% acrylamide) (Schägger and von Jagow, 1987).…”