| Assay Method Information | |
| | CDK protein kinase inhibition by the ADP-Glo Assay |
| Description: | This ADP-Glo assay measures ADP formed from a kinase reaction and indirectly quantifies the phosphorylation of peptide substrates by the CDK/cyclin protein complexes. The typical assays were carried out in Nanosyn by combining kinase/cyclin complexes, substrates, compounds, and cofactors (ATP and Mg2+) in a well of a 384-well microtiter plate (Coming 3824) and incubating at 22° C. (Table 4). At the end of the incubation, the reaction is quenched, and conversion is detected by luminescence using the Promega ADP-Glo™ Kinase Assay kit (catalog #V9102). Briefly, 5 μL of 1× enzyme and substrate buffer (or control), 50 nL of 100× compound, and 0.5 μL of 10 mM ATP were sequentially added to a well of a 384-well plate. The Final assay buffer mixture contains: 100 mM HEPES, pH 7.5, 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 5 mM MgCl2, 10 μM Sodium Orthovanadate, 10 μM Beta-Glycerophosphate, 1000 μM Ultra Pure ATP (provided in the Promega ADP-Glo™ Kinase Assay kit), and 1% DMSO (from compound). Upon completion, 5 μL of the ADP Glo reagent was added to each well and the plate was further incubated at room temperature for 45 min. 10 μL of the Kinase Detection Reagent was subsequently added to each well. After 10 min of incubation, the plate was read and analyzed on the BioTek Synergy microplate reader using BioTek Gen5 software. |
| Affinity data for this assay | |
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