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| | DELFIA® Assay |
| Description: | In vitro CHK2 kinase activity was measured in a DELFIA® assay that monitors phosphorylation of a CDC25C peptide using a specific phospho antibody. The enzyme reaction was carried out in 96-well polypropylene plates (Greiner). The reaction mix (total volume 25 μL) contained enzyme and peptide mix (15 μL) (containing CHK2, prepared in-house, 1 nM; Biotin-KKKVSRSGLYRSPSMPENLNRPR, 1 μM), ATP (30 μM, 5 μL) and either DMSO (2.5%) or test compound (5 μL) diluted to a give a range of concentrations (0-100 μM in 2.5% DMSO, final concentrations) in assay buffer (40 mM HEPES (pH 7.4), 40 mM KCl, 2 mM MgCl2, 10 mM DTT and 0.02% Tween 20). The reaction mixture was incubated for 30 minutes at room temperature and stopped by the addition of buffer (125 μL) containing 40 mM EDTA, 0.05% Tween 20, 0.1% BSA in TBS (10× concentrate, Sigma). An aliquot (100 μL) of the reaction mix was transferred to a black neutravidin-coated 96-well plate (Perbio) and incubated for 1 hour on a shaker (Titertek, Flow Laboratories) at room temperature. The plates were washed four times with wash buffer (25 mM Tris (pH 8), 150 mM NaCl and 0.1% Tween 20) (WellWash4, Thermo Life Sciences) and incubated for 1 hour as before with antibody mix (100 μL) consisting of anti-phospho CDC25C (diluted 1/4000 equivalent to 0.35 nM-1.25 nM, #9528, Cell Signalling Technology) and europium-labelled anti rabbit IgG, (0.3 μg/mL, AD0105, PerkinElmer Life Sciences) diluted in DELFIA® assay buffer (PerkinElmer Life Sciences). The plates were washed a further four times with wash buffer before the addition of enhancement solution (100 μL/well, PerkinElmer Life Sciences). The plate was read on a Victor2 1420 multi label counter (PerkinElmer Life Sciences) using a time-resolved measurement mode reading fluorescence at 615 nM. |
| Affinity data for this assay | |
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