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.