| Assay Method Information | |
| | Determination of Inhibitor Potency Vs. CHK1 in Caliper Assay Format |
| Description: | CHK1 kinase activity was measured in a microfluidic assay that monitors the separation of a phosphorylated product from its substrate. The assay was run on an EZ Reader II (Caliper Life Sciences Ltd, Runcorn, UK) using separation buffer (#760367 Caliper LS) containing CR-8 (500 nM, #760278, Caliper LS). An ECHO 550 (Labcyte Inc ) acoustic dispenser was used to generate duplicate 8 pt dilution curves directly into 384 polypropylene assay plates (Greiner Bio-One, Gloucestershire, UK). For each test compound a 50 μM stock concentration in 100% DMSO was used. The total amount of DMSO dispensed per well was 250 nL to give a final assay concentration of 2.5% DMSO and test compound concentrations in the range 0.5-1000 nM. To this assay plate, 6 μL CHK1 (2 nM final concentration, in-house protein preparation), 2 μL peptide 10 (5-FAM-KKKVSRSGLYRSPSMPENLNRPR-COOH (SEQ ID NO: 1), 1.5 μM final concentration, #760354 Caliper LS) and 2 μL ATP (90 μM final concentration) all diluted in kinase buffer (HEPES 50 mM, NaN3 0.02%, BSA 0.01%, sodium orthovanadate 0.1 mM, DTT 1 mM, MgCl2 2 mM, Tween 20 0.1%) were added. The plate was sealed and centrifuged (1 minute, 1000 rpm) before incubation for one hour at room temperature. The reaction was stopped by the addition of separation buffer (90 μL). |
| Affinity data for this assay | |
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