Br. Herbert et al., IMPROVED PROTEIN SOLUBILITY IN 2-DIMENSIONAL ELECTROPHORESIS USING TRIBUTYL-PHOSPHINE AS REDUCING AGENT, Electrophoresis, 19(5), 1998, pp. 845-851
Citations number
15
Categorie Soggetti
Biochemical Research Methods","Chemistry Analytical
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.