| Assay Method Information | |
| | Biological Assay |
| Description: | The Polθ ATPase assay was used to evaluate inhibitors of Polθ ATPase activity, in vitro. Experiments were performed using a truncated Polθ protein (Polθ-Hel containing the helicase domain (67-894), ATP and a DNA single strand Oligo (5′-CTGTCCTGCATGATG-3′) in the Polθ assay buffer (20 mM Tris-HCl (pH 8.8), MgCl2 5 mM, tween20 0.01%, NP40 0.01%, BSA 0.01%, DTT 1 mM). 2 μL of assay buffer containing Polθ-Hel protein (0.5 nM) and DNA substrate (500 nM) was transferred into assay ready plates, containing 0.04 μL of compounds diluted in DMSO. After a 30 minutes incubation at 23° C. in the dark, the reaction was triggered by adding 2 μL of ATP (60 μM), in assay buffer. After 60 minutes at 23° C., 4 μl of ADP-Glo™ Reagent (Glo 1) was added followed by 40 minutes of incubation, then 8 μl of ADP-Glo™ kinase Detection Reagent (Glo 2) followed by 60 minutes of incubation at 23° C. Luminescence was then measured using Tecan F200 infinite plate reader. Raw data were analysed using GeneData to generate IC50 values. |
| Affinity data for this assay | |
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