| Assay Method Information | |
| | CDK2/Cyclin E1 Full length ADP-Glo Kinase Assay |
| Description: | The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition and IC50 values) of small molecule inhibitors by using a Luminescent based ADP-Glo assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP. ADP-Glo assay monitors ADP producing biochemical reactions. ADP-Glo is performed in 2 steps upon completion of kinase reaction: a combined termination of kinase reaction and depletion of remaining ATP in the first step, and conversion of generated ADP to ATP and the newly produced ATP to light output using luciferase/luciferin reaction in the second step. The luminescent signal generated is proportional to the ADP concentration produced and is correlated with the kinase activity. CDK2/Cyclin E1 was purchased from Eurofins (Cat 14-475M). Typical reaction solutions (10 uL final reaction volume) contained 2% DMSO (±inhibitor), 10 mM MgCI2, 1 mM EGTA, 0.05% BSA, 2 mM DTT, 20 uM ATP (ATP Km=64.78 uM), 0.01% Brig-35, 0.75 uM substrate, and 0.328 nM wild-type full length CDK2/Cyclin E1 enzyme complex in 50 mM HEPES buffer at pH 7.5. The assay was initiated with the addition of ATP-containing substrate solution, following a 30-minute pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 90 minutes at room temperature by the addition of 10 uL of ADP-GLO Reagent. After a 90 minute incubation, 20 uL of Kinase Detection Reagent was added. Samples were incubated for 40 minutes, after which plate well luminescence was measured on a Envision microplate reader. The IC50 determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the 4 parameters IC50 equation. |
| Affinity data for this assay | |
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