| Assay Method Information | |
| | ADP-Glo assay |
| Description: | PolQ ATPase activity was determined by ADP-Glo assay. 10-point dilution series of compounds were used in a 384 well format for the inhibition assays. PolQ (1-899) (1 nM) in assay buffer (20 mM Tris HCl (pH 8.0), 80 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.01% BSA, 0.01% Tween, 5% glycerol) was transferred to the test wells (20 uL), except the low control wells (20 μL of assay buffer was added to the low control wells). The plate was then incubated at room temperature for 30 min. An equal volume (20 μL) of 100 μM ATP, 150 nM ssDNA containing 50 thymine bases in assay buffer was added to all the test wells. The plates were covered and left to incubate for 60 min. at room temperature before the addition of the ADP Glo detection reagents. After 60 min. incubation, transfer 5 μL reaction mix to another 384-well plate and add 5 μL ADP Glo and plates incubated for 60 min. before addition of 10 μL kinase detection reagent. After the addition of the kinase detection reagent, the plates were covered and incubated for 60 min. |
| Affinity data for this assay | |
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