| Literature DB >> 23954627 |
Cody W Lewis1, Ryan G Taylor, Philip M Kubara, Kris Marshall, Laurent Meijer, Roy M Golsteyn.
Abstract
We developed a quantitative method to measure the activity of cyclin-dependent kinases (Cdks) by western blotting, without radioisotopes. We prepared a recombinant protein substrate based upon the natural Cdk1 substrate, PP1Cα. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the Km of ATP binding to Cdk1 to be 3.5 μM. We then measured Cdk1 activity in cell extracts from interphase or mitotic cells, and demonstrated that previously identified Cdk inhibitors could be detected by this assay. Our data show that we have a safe, reliable assay to identify Cdk1 inhibitors and measure Cdk1 activity. CrownEntities:
Keywords: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; CPT; Cdk1; Cdk2; DMSO; DTT; EGTA; HEPES; IPTG; Mitosis; PBS; Protein phosphatase 1Cα; Tissue culture; camptothecin; cyclin dependent kinase 1; cyclin dependent kinase 2; dimethyl sulfoxide; dithiothreitol; ethyleneglycol-bis (beta-aminoethylether)- N,N′-tetraacetic acid; isopropyl β-d-1-thiogalactopyranoside; phosphate-buffered saline
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Year: 2013 PMID： 23954627 DOI： 10.1016/j.febslet.2013.08.003
Source DB: PubMed Journal: FEBS Lett ISSN： 0014-5793 Impact factor: 4.124