Assay Method Information
Assay Name:  Radiometric Protein Kinase Assay
Description:  A radiometric protein kinase assay (33PanQinase® Activity Assay) was used for measuring the kinase activity of the two protein kinases (CDK12/CDK7). All kinase assays were performed in 96-well FlashPlates™ from PerkinElmer (Boston, MA, USA) in a 50 μL reaction volume.The reaction cocktail was pipetted in four steps in the following order:20 μL of assay buffer (standard buffer)5 μL of ATP solution (in H2O)5 μL of test compound (in 10% DMSO)10 μL of substrate/10 μL of enzyme solution (premixed)[2102]For the CDK7 assay, a reaction mixture (50 μL) containing the following components was prepared:70 mM HEPES-NaOH (pH 7.5);sodium orthovanadate (3 μM);PEG-20000 (50 μg/mL);DTT (1.2 mM);MgCl2 (3 mM);MnCl2 (3 mM);purified human CDK7/CycH/MAT1 (3.3 nM—Lot 02);RBER-CHKtide substrate (40 μg/mL—Lot 106);ATP (3 μM);[γ-33P]-ATP (approx. 8×105 cpm per well); andtest compound at the appropriate concentration such that the final concentration of DMSO was 10% w/w.[2114]For the CDK7 assay, a reaction mixture (50 μL) containing the following components was prepared:70 mM HEPES-NaOH (pH 7.5);sodium orthovanadate (3 μM);PEG-20000 (50 μg/mL);DTT (1.2 mM);MgCl2 (3 mM);MnCl2 (3 mM);purified human CDK12 wt/CycK (14.7 nM—Lot 02);RBER-IRStide substrate (40 μg/mL—Lot 036);ATP (0.3 μM);[γ-33P]-ATP (approx. 8×105 cpm per well); andtest compound at the appropriate concentration such that the final concentration of DMSO was 10% w/w.The reaction mixture was incubated at 30° C. for 60 min and then stopped by the addition of 2% (v/v) H3PO4. Plates were aspirated and washed two times with 200 μL 0.9% (w/v) NaCl. Incorporation of 33Pi was determined with a microplate scintillation count (Microbeta, Wallac).The residual activities for each concentration and the compound IC50 values were calculated using Quattro Workflow V3.1.1 (Quattro Research GmbH, Munich, Germany).
Affinity data for this assay
 
If you find an error in this entry please send us an E-mail