SBE13

Kinase experiment:

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].

References:

[1]. Keppner S, et al. Identification and validation of a potent type II inhibitor of inactive polo-like kinase 1. ChemMedChem. 2009 Nov;4(11):1806-9.
[2]. Keppner S, et al. Fate of primary cells at the G /S boundary after polo-like kinase 1 inhibition by SBE13. Cell Cycle. 2011 Feb 15;10(4):708-20. Epub 2011 Feb 15.
[3]. Keppner S, et al. Biological impact of freezing Plk1 in its inactive conformation in cancer cells. Cell Cycle. 2010 Feb 15;9(4):761-73. Epub 2010 Feb 16.