CHK1 kinase activity assay.
Mammalian CHK1 is a Ser/Thr effector kinase that plays critical roles in the DNA damage-activated cell cycle checkpoint signaling pathway downstream of ATR (ATM and Rad3-related protein kinase). This chapter is focused 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; and (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 CHK2 activity assay, except that the CHK2 antibody will replace the CHK1 antibody.
Wang
Ya
Y
Department of Radiation Oncology, Kimmel Cancer Center of Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA.
Wang
Hongyan
H
D000911:Antibodies, Monoclonal / Q000276:immunology; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D016195:G2 Phase; D006801:Humans; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011233:Precipitin Tests / Q000379:methods; D011494:Protein Kinases / Q000032:analysis*; D016196:S Phase; D020687:cdc25 Phosphatases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; D010761:Phosphorus Radioisotopes; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases