| Assay Method Information | |
| | In Vitro Inhibitory Activity Against ATR Kinase |
| Description: | A 50 ng/μL ATR stock solution was diluted with a kinase buffer (50 mM HEPES, 10 mM MgCl2, 2 mM DTT, 1 mM EGTA, 0.01% Tween 20), and 6 μL of 1.67×, 0.0835 ng/μL working solution was added to each well (final concentration: 0.05 ng/μL). Different compounds dissolved in DMSO were added to wells using a nanoliter pipettor, such that the final concentrations of the compounds were 1000 nM-0.24 nM and the concentrations in positive wells were 100 nM-0.024 nM (4-fold gradient, 7 concentrations in total). Meanwhile, a blank control well (containing no enzyme) and a negative control well (containing enzyme, with the vehicle DMSO added) were set. After the enzyme had reacted with the compound or vehicle for 30 min, a 5×, 50 μM ATP (final concentration: 10 μM) prepared with the kinase buffer and a 5×, 0.5 μM substrate (final concentration: 0.1 μM, U Light-poly GT) were 1:1 mixed, and the mixture was added to wells at 4 μL/well. After the plate was sealed with a sealing film and the plate was incubated at room temperature for 2 h, 5 μL of 4×, 40 mM EDTA (final concentration: 10 mM) was added to each well. After 5 min of incubation at room temperature, 5 μL of a 4×, 8 nM assay reagent (final concentration: 2 nM, Ep-anti-phospho-tyrosine antibody) was added to each well. The plate was incubated at room temperature for 1 h. Plate reading was performed on a PE instrument (excitation: 320 or 340 nm; emission: 665 nm), and four-parameter fitting and IC50 calculations were performed. |
| Affinity data for this assay | |
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