SBE13

To assay Plk1 kinase activity, cells are lysed after 13 h release in the presence of SBE13 after double thymidine block and kinase is immunoprecipitated from lysates using antibodies. In brief, for each immunoprecipitation 800 μg of total protein are incubated with Plk1 antibody cocktail (1.5 μg) for 2 h at 4°C on a rotator. Immunoprecipitated protein is collected using Protein A/G Agarose beads. Plk1 immunoprecipitates are incubated with casein (1 μg) and with [γ-³²P]ATP (1 μCi) for 30 min at 37°C in kinase buffer. Products from the kinase assays are fractionated on 10 % bis-tris-polyacrylamide gels, and phosphorylated substrate is visualized by autoradiography after an exposure of 12-36 h. Equal amounts of immunoprecipitates are subjected to Western blot analysis to confirm equal loading of Plk1 protein in kinase reactions^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.