IMPROVED PROTEIN SOLUBILITY IN 2-DIMENSIONAL ELECTROPHORESIS USING TRIBUTYL-PHOSPHINE AS REDUCING AGENT

In this study, dithiothreitol was replaced by tributyl phosphine as th e reducing agent in both the sample solution for the first-dimensional isoelectric focusing and during the immobilised pH gradient (IPG) equ ilibration procedure. Tributyl phosphine improves protein solubility d uring isoelectric focusing, which results in shorter run times and inc reased resolution. Tributyl phosphine is nonionic and thus does not mi grate 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 focu sing and decreased horizontal streaking on the subsequent second-dimen sion gel. The use of tributyl phosphine in the equilibration step allo ws the procedure to be simplified, incorporating reduction and alkylat ion 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.