title: A western blot assay to measure cyclin dependent kinase activity in cells or in vitro without the use of radioisotopes.
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.
authors: Cancer Cell Laboratory, Department of Biological Sciences, University of Lethbridge, 4401 University Dr, Lethbridge, AB T1K 3M4, Canada.
authors: Cody W
authors: CW
authors: Lewis
authors: Ryan G
authors: RG
authors: Taylor
authors: Philip M
authors: PM
authors: Kubara
authors: Kris
authors: K
authors: Marshall
authors: Laurent
authors: L
authors: Meijer
authors: Roy M
authors: RM
authors: Golsteyn
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western / Q000379:methods* / Q000592:standards; D016203:CDC2 Protein Kinase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019073:HT29 Cells; D006801:Humans; D061986:MCF-7 Cells; D008938:Mitosis / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D016203:CDC2 Protein Kinase; D054645:Protein Phosphatase 1
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