| Assay Method Information | |
| | ADP-Glo Kinase Assay |
| Description: | ADP-Glo assay kit was purchased from Promega and the CDK9/CycT1 protein and PDKtide substrate were purchased from SignalChem. Compounds of Structure (I) are dosed in 10-dose IC50 mode in duplicate with 3-fold serial dilution starting at 100 μM. The reaction is started by pre-incubating 4 μL of protein (20 nM), 2 μL of substrate (100 μM), and 2 μL of serial diluted drug in 1× assay buffer (50 mM HEPES, 3 mM MgCl2, 3 mM MnCl2, and 1 mM DTT) for 30 minutes. After pre-incubating, 4 μL of ATP (1 μM) is added to each well and incubated for an additional 90 minutes. The ADP-Glo Reagent (10 μL) and Kinase Detection Reagent (20 μL) are added to each well and incubated as recommended by Promega. The reaction is quantified by measuring luminescence and the IC50 is calculated using GraphPad Prism software. |
| Affinity data for this assay | |
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