CMK

Kinase experiment:

For Cdc5 kinase assays, Spc72-TAP is isolated from cell extract by pulldown with IgG sepharose or rabbit IgG coupled to M-270 epoxy dynabeads and incubated in kinase buffer (25 mM HEPES, pH 8.0, 60 mM KCl, 15 mM MnCl2, 100 μg/mL BSA, 80 nM microcystin, 1mM DTT, 100 μM 200 μCi/mL [γ-32P]ATP) in the absence or presence of 100 ng purified baculovirus expressed 6xHis-Cdc5. 32P incorporation is visualized on a Typhoon PhosphorImager, and images are processed using ImageQuant software.

References:

[1]. Snead JL, et al. A coupled chemical-genetic and bioinformatic approach to Polo-like kinase pathway exploration. Chem Biol. 2007 Nov;14(11):1261-72.