Analysis of phosphorylation of protein synthesis initiation factor eIF-2 by two-dimensional gel electrophoresis.

Abstract

We describe a method for examining the state of phosphorylation of initiation factor eIF-2 in reticulocyte lysates. The procedure involves incubation of the lysate with iodo[1-14C]acetate in 8.5 M urea, and fractionation of the labelled proteins by two-dimensional acrylamide gel electrophoresis. This approach has been used to show that haem deficiency, double-stranded RNA, and oxidised glutathione, which all inhibit the initiation of protein synthesis in an analogous manner, all cause a net increase in the level of phosphorylated eIF-2 in the complete lysate protein synthesis system.