| Assay Method Information | |
| | Human Chk1 HTRF (Homogeneous Time Resolved Fluorescence) Enzyme Assay |
| Description: | Table 20: 2 μL enzyme/substrate solution (1 nM Chk-1 (Carna Bioscience #02-177), in solution with 30 μM ATP, 4 μM STK1 S1 (Cisbio #62ST1PEB) in assay buffer (20 mM HEPES pH7.5, 500 mM NaCl2, 0.1% BGG, 1 mM DTT, 0.005% Tween-20)) and different concentrations of compounds were incubated at room temperature for 30 minutes in 384-well plates (proxiplate-384 plus, Perkin Elmer cat #6008289). MgCl2 cofactor solution was added (2 μL, 20 mM) and incubation continued at room temperature for 30 minutes. 4 μL kit detection reagent (Cisbio #62ST1PEB) was added and incubation continued for 60 minutes at room temperature. Plates were read on the PHERAstar FS using TR-FRET technology, channel A: 337/665, channel B: 337/620. % effects of a compound at a given concentration was calculated based on inhibition produced in the DMSO control wells and inhibited control wells control wells in each assay plate. Compound IC50s were determined with a four-parameter logistic dose response equation using 11 point dose response curves, analysing within the Dotmatics platform. |
| Affinity data for this assay | |
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