| Assay Method Information | |
| | CHK-1 Kinase Assay |
| Description: | IC50 values of compounds for inhibition of CHK-1 kinase or percent inhibition at defined concentrations were determined by Z-LYTE™ based TR-FRET assay. Kinase reactions were performed in a 10 μL volume in low-volume 384-well plates. The concentration of substrate (Ser Thr 19) was maintained at 2 μM in the assay, and the kinase reaction buffer consisted of 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT. Serially diluted compounds (3-fold) in DMSO (0.5% in final reaction) were incubated with cocktail of CHK-1 kinase (5 ng/ml; Cat #P3040, ThermoFisher), substrate Ser Thr 19, (2 μM; Cat #PV4529, ThermoFisher), ATP (53 μM; Cat #PV3227, ThermoFisher) and buffer. Kinase reactions were allowed to proceed for 1 hour at room temperature after which 5 μL of a 1:256 dilution of Development Reagent A was added. The plate was allowed to incubate at room temperature for 60 minutes before being read on a BMG Pherastar plate reader using the Z-LYTE™ filter block for Z-LYTE™ TR-FRET. |
| Affinity data for this assay | |
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