The importance of the DDR is underscored by the fact that failure to activate DNA damage checkpoints raises genomic instability and can cause a selection of diseases. By comparison, Chk1 and ATR are necessary for mammalian cell viability, and knockout mice for these proteins show embryonic Cathepsin Inhibitor 1 lethality. The essential roles of Chk1 in the cell remain unclear, for the reason that hardly any substrates of Chk1 have now been identified to date. As hundreds of protein kinases are secured by the human genome, all of which use as their co factor ATP, and because tens of thousands of potential phosphorylation sites have been identified in human proteins, it’s been complicated to determine kinase substrate relationships. Recognition of such pairs is generally based on the investigator making an informed guess, accompanied by in vitro kinase assays and in vivo evidence with phospho specific antibodies. The identity of the kinase is then further confirmed by the use of particular kinase inhibitors and/or small interfering RNA mediated kinase destruction. Screening for large numbers Cellular differentiation of protein kinase substrates has proven more challenging, though current antibody based screens have identified countless putative ATM and ATR substrates. As such tests require the previous identification of internet sites of substrate phosphorylation and corresponding antibodies that specifically recognize these phosphorylated motifs, these methods are unfortuitously perhaps not feasible for kinases such as Chk1 that have few known targets, that share phosphorylation motifs with other kinases and/or lack a very specific target motif. Chemical genetics uses Checkpoint inhibitor small molecule modulators of protein and nucleic acid activities to elucidate cellular features of the targets. Somewhat, Shokat and co workers are suffering from a chemical genetics system to modulate the action of a protein kinase by mutating an amino-acid residue in its ATP binding pocket, allowing the resulting kinase known as an analogue sensitive kinase to support a bulky ATP analogue. This altered ATP binding pocket enables the specific inhibition of the as kinase in vivo by using specific cell membrane permeable, nonhydrolysable ATP analogues. More recently, new methods to determine in vitro substrates of as kinases have been developed that marked ATP analogue in cell extracts and include the use of a hydrolysable. This latter approach is successfully applied to the identification of new substrates of protein kinases such as CDK1/ CyclinB, CDK7, and CDK2/CyclinA. Here, by making use of this method to Chk1, we establish 268 phosphorylation sites in proteins, thus providing for the very first time a fair set of putative Chk1 substrates. as Chk1 identifies phosphorylation websites and new in vitro substrates A new, elegant method designed to identify substrates of an as kinase involves using an ATP analogue carrying a thio phosphate group.