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A western blot assay to measure cyclin dependent kinase activity in cells or in vitro without the use of radioisotopes
Author(s) -
Lewis Cody W.,
Taylor Ryan G.,
Kubara Philip M.,
Marshall Kris,
Meijer Laurent,
Golsteyn Roy M.
Publication year - 2013
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2013.08.003
Subject(s) - cyclin dependent kinase 1 , cyclin dependent kinase , western blot , kinase , blot , microbiology and biotechnology , recombinant dna , mitosis , chemistry , cyclin dependent kinase 2 , biochemistry , biology , cell cycle , protein kinase a , cell , gene
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 K m 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.