Generation of phosphorylation state-specific SRC-class kinase antibodies for analysis of kinase activation

Protein phosphorylation is a major molecular mechanism by which cellular fu nction is regulated. In order to accomplish rapid and specific biochemical changes via phosphorylation, the activity of a protein kinase must be dynam ically regulated. Historically, the activity of each protein kinase has bee n analyzed using a unique in vitro biochemical assay with a specific substr ate and detection procedure. These assays require the use of radioactivity and are often labor intensive. Upon activation, most protein kinases autoph osphorylate. Thus, a technical approach to detect changes in kinase activit y is to measure autophosphorylation. The purpose of this protocol is to pro vide a detailed stepwise procedure for measuring the regulation of Src-clas s kinase activity using phosphorylation state-specific antibodies. Antibodi es to a phosphorylated peptide derived from the autophosphorylation site of Src-family kinases are developed and affinity purified. The purified antib odies are used to analyze the regulation of Src and Fyn activity in a mouse muscle cell line. It is anticipated that the utility of these phosphorylat ion state-specific antibodies will ultimately result in the development of similar antibodies useful for analyzing the activity of many different kina ses. (C) 2000 Elsevier Science B.V. All rights reserved.