Cdk2 kinase assay

Cdk2 kinase assay reactions were setup according to manufacturer protocol of ADP-Glo kinase assay (Promega). Basically, each 25 μl reaction contains 1x kinase buffer (40 mM Tris-HCl pH=7.4, 20 mM MgCl2, 0.1 mg/ml BSA, and 1 mM DTT), 1 μg Histone H1 (Sigma), 100 μM ultra pure ATP (Promega). Each reaction was then added with 1000, 500, 250, 125, 62.5, 31.25, 15.625, or 0 ng Cdk2^WT-His6 or Cdk2^160TAG-His6. Biological triplicates were performed at each concentration. Reactions were then incubated at 30°C for 40 min, cooled down at RT for 10 min, and mixed with 25 μl ADP-Glo reagent. After 40 min incubation at RT, 50 μl Kinase Detection Reagent was further added to each reaction and incubated at RT for 40 min. The luminescence for each reaction was then measured by Pherastar Plate Reader.