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

Protein phosphorylation is a major molecular mechanism by which cellular function is regulated. In order to accomplish rapid and specific biochemical changes via phosphorylation, the activity of a protein kinase must be dynamically regulated. Historically, the activity of each protein kinase has been analyzed using a unique in vitro biochemical assay with a specific substrate and detection procedure. These assays require the use of radioactivity and are often labor intensive. Upon activation, most protein kinases autophosphorylate. Thus, a technical approach to detect changes in kinase activity is to measure autophosphorylation. The purpose of this protocol is to provide a detailed stepwise procedure for measuring the regulation of Src-class kinase activity using phosphorylation state-specific antibodies. Antibodies to a phosphorylated peptide derived from the autophosphorylation site of Src-family kinases are developed and affinity purified. The purified antibodies 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 phosphorylation state-specific antibodies will ultimately result in the development of similar antibodies useful for analyzing the activity of many different kinases.