| Assay Method Information | |
| | Polθ Polymerase Domain Enzyme Inhibition |
| Description: | In the assay, Polθ interacts with the substrates (deoxy thymidine triphosphate (dTTP) (Sigma, product code T0251) and a DNA template with a 17-nucleotide overhang (Eurogentec, custom creates order), and PPi is formed due to the polymerase reaction. This free PPi interacts with AMP, to generate the ATP used in the luciferase reaction in the production of light. The test compound competitive binding inhibits the polymerase reaction, resulting in the loss of luminescence.The assay was performed as follows with all reagent additions carried out using a CERTUS FLEX liquid dispenser workstation:Test compound (15 nL) was acoustically dispensed into Greiner 1536 well white small volume medium bind assay plates.1X assay screening buffer (50 mM Tris pH7.5, 5 mM MgCl2, 0.01% v/v Pluronic F127, 2 mM DTT, 0.05 mg/ml BSA) is prepareddTTP/DNA (1.5 μL) was dispensed of into each of the wells followed by 1.5 μL of Polθ, the plates are covered and the reaction is allowed to progress for 20 minutes at room temperature.To quench, TFA (0.5 μL) was dispensed into the wells, then 2.0 μL of PPiLight reagent was dispensed into each well and incubated at room temperature for 1 hour.Plates were then read on an EnVision plate reader for luminescence 400-700 nm.Compounds were dosed directly from a compound source plate containing serially diluted compounds (4 wells containing 10 mM, 0.1 mM, 1 μM and 10 nM respectively) to an assay microplate using a labcyte ECHO 550 liquid handler. The ECHO 550 using acoustic technology to transfer between microplates of DMSO compound solutions, with the system being able to be programmed to transfer small nL volumes of compounds from different source plate wells to give serial dilutions for the compounds to be tested and backfilled to normalise the DMSO concentration across the dilution range. |
| Affinity data for this assay | |
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