| Assay Method Information | |
| | CDK6/Cyclin D3 ADP-Glo Kinase Assay |
| Description: | The purpose of the CDK6/Cyclin D3 assay is to evaluate the inhibition (% inhibition and IC50 values) in the presence of small molecule inhibitors by using a Luminescent based ADP-Glo assay. CDK6/Cyclin D3 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. CDK6/Cychin D3 was purchased from Carna. Typical reaction solutions (10 uL final reaction volume) contained 2% DMSO (±inhibitor), 10 mM MgCI2, 1 mM EGTA, 0.05% BSA, 2 mM DTT, 100 uM ATP (ATP Km=291.7 uM), 0.01% Brig-35, 0.75 uM substrate, and 5 nM wild-type CDK6/Cyclin D3 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 μL of ADP-GLO Reagent. After a 90-minute incubation, 20 μL 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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