Production of phosphorylation state-specific antibodies.

Publisher Summary This chapter discusses the production of phosphorylation state-specific antibodies. The ability to detect and to quantitate changes in the state of phosphorylation of specific substrate proteins is of great utility in the study of their functional significance. Standard methods for measuring the state of protein phosphorylation in intact cell preparations utilize prelabeling with [ 32 P]P i or back phosphorylation. The degree of sensitivity and selectivity afforded by immunochemical methodology makes it an attractive alternative for detecting changes in the state of phosphorylation of specific proteins. The methods used for these proteins have general application in the production of phosphorylation state-specific antibodies to substrates whose sites of phosphorylation have been established.