Assay Method Information

Assay Name:  Protein Kinase Assay
Description:  We incubated human recombinant CDK2/cyclin A with small molecules at the indicatedconcentrations in buffer A (Tris HCl (25 mm, pH 7.5), MgCl2 (10 mm), EGTA (1 mm), DTT (1 mm), heparin (50 mg/mL)) containing histone H1 (1 mg/mL), ATP (15 mm) and [g-33P]ATP (15 mm, 3,000 Cimmol^-1; 10 mCi/mL) in a final volume of 30 mL. After 30 min incubation at 30 C , we stopped the reaction by using a MicroBeta FilterMate-96 Harvester and P81 phosphocellulose papers , which we washed in phosphoric acid (1 %). We added scintillation fluid, and we measured the radioactivity in a counter with microplate robotic stackers. We calculated kinase activity as picomoles of phosphate incorporated during the 30 min incubation. We evaluated percentage kinase activity as the amount compared to that measured in the absence of inhibitors.
Affinity data for this assay
 

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