| Assay Method Information | |
| | Biochemical Pol Theta (1-894) ATPase IC50 Assay |
| Description: | Enzymatic reactions of Pol Theta (1-894) were performed using ADP-Glo assay with ssDNA substrate. Pol Theta (1-894) was expressed in baculovirus expression system. The inhibition activities testing for the compound disclosed herein were carried out at room temperature in assay buffer containing 40 mM Tris pH 7.5, 20 mM MgCl2, 1 mM DTT, 0.01% BSA and 0.05% Tween-20. Compounds in DMSO were dispensed into wells of a black 384 well plate (Corning 4514) using D300e digital dispenser (Tecan). The ranges of compounds final concentration were 2.62 nM˜10000 nM. 3 μL 2× enzyme solution containing 12.5 nM enzyme was added to wells. After incubation for 1 hour, 3 μL 2× substates solution containing 0.124 μM ssDNA (5′-ACT CGT CTC TAG CTT TTT-3′) and 444 μM ATP was added to the wells to initiate reaction. After 1 hour reaction, 5 μL ADP-Glo reagent (Promga V9102) was added and incubated for 40 minutes. 10 μL Kinase Detection reagent was added and incubated for 40 minutes. Luminescence was measured on a microplate reader (PHERAstar FSX, BMG labtech). The IC50 values were calculated based on inhibition of enzyme activity in the presence of increasing concentrations of compounds. |
| Affinity data for this assay | |
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