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

Ben R. Herbert, Mark P. Molloy, Andrew A. Gooley, Bradley J. Walsh, Warren G. Bryson, Keith L. Williams*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

209 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)845-851
Number of pages7
JournalElectrophoresis
Volume19
Issue number5
DOIs
Publication statusPublished - May 1998

Keywords

  • immobilized pH gradients
  • solubility
  • tributyl phosphine
  • two-dimensional polyacrylamide gel electrophoresis

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