Product Pathways - Kinases
Chk1 Kinase #7359
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Description
Purified recombinant full-length human Chk1 (Met1-Thr476) kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human Chk1 (Met1-Thr476) (GenBank Accession No. NM_001274) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-Chk1 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. Chk1 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: CHKtide 400 ng/μL, and variable amounts of Chk1 kinase.
Quality Control
The theoretical molecular weight of the GST-Chk1 fusion protein is 82 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. Chk1 kinase activity was determined using a radiometric assay [Fig.2].
Background
Chk1 kinase acts downstream of ATM/ATR kinase to play an important role in DNA damage checkpoint control, embryonic development and tumor suppression (1). Activation of Chk1 involves phosphorylation of Ser317 and Ser345 and occurs in response to blocked DNA replication and certain forms of genotoxic stress (2). Chk1 is also phosphorylated at Ser280 and Ser296 following DNA damage. Activated Chk1 can inactivate cdc25C via phosphorylation at Ser216, blocking the activation of cdc2 and transition into mitosis (3). Chk1 can also phosphorylate p53 at Ser20 in vitro (4).
- Martinho, R.G. et al. (1998) EMBO J. 17, 7239-7249.
- Zhao, H. et al. (2001) Mol. Cell. Biol. 21, 4129-4139.
- Zeng, Y. et al. (1998) Nature 395, 507-510.
- Shieh, S. et al. (2000) Genes Dev. 14, 289-300.
Application References
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Companion Products
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
