CHK1 kinase activity assay

Abstract

Mammalian CHK1 is a Ser/Thr kinase that plays a critical role in the DNA damage-activated cell cycle checkpoint signaling pathway downstream of ATR (ATM and Rad3 related protein kinase). This chapter focuses on describing an assay to measure CHK1 activity in vitro. The basic mechanism of this assay is to observe the phosphorylated levels of a fragment of CDC25C containing the site that can be phosphorylated by CHK1 in vitro. This assay includes five major steps: (1) preparing extracts from the control or treated cells, (2) preparing substrate, (3) immunoprecipitating CHK1 protein from the cells, (4) assembling the kinase assay, (5) analyzing the phosphorylated level of the substrates by CHK1. Besides CHK1, CHK2 is another important checkpoint regulator that responds to DNA damage. Because CHK1 and CHK2 share some substrates such as CDC25C in vitro, this assay could also be used for a CHK2 activity assay, except that the CHK2 antibody will be replaced by the CHK1 antibody. © 2012 Springer Science+Business Media New York.