| Assay Method Information | |
| | ATPase Assay |
| Description: | POLθ-mediated ATP hydrolysis was measured using the ADP-Glo Kinase Assay (Promega Corporation, Madison, WI). Reactions were performed in white 384-well small-volume microplates (Greiner Bio-One, Frickenhausen, Germany) using a total volume of 15 μL. The ATPase reaction consisted of 1 nM biotinylated Avi-POLθ[2-894], 5 nM of a 50 nucleotide poly-thymine repeat single-stranded DNA (PolyT(50) ssDNA), and 100 μM ATP in 5 μL of assay buffer (50 mM Tris, pH 7.5, 80 mM KCl, 10 MgCl2, 1 mM DTT, 5% glycerol, 0.01% BSA, 0.01% Tween-20). Inhibition of POLΘATPase activity was determined by preparing 11-point serial dilutions of the test compounds in 100% DMSO, then transferring 30 nL of the compounds into the ATPase reaction using an Echo acoustic liquid handler (Beckman Coulter, Brea, CA). DMSO-treated wells containing either all of the reaction components (1 nM biotinylated Avi-POLθ[2-894], 5 nM PolyT(50) ssDNA, 100 μM ATP) or with assay buffer substituted for the biotinylated Avi-POLθ[2-894](0 nM biotinylated Avi-POLθ[2-894], 5 nM PolyT(50) ssDNA, 100 μM ATP) were used to define the maximum and minimum response in the assay. Following a one-hour incubation at ambient temperature, 5 μL of ADP-Glo Reagent was added to the ATPase reaction, and this mixture was allowed to incubate at ambient temperature for an additional hour. Lastly, 5 μL of Kinase Detection Reagent was added, followed by a third one-hour incubation at ambient temperature. The reagent concentrations listed represent the amounts only in the 5 μL ATPase reaction volume. |
| Affinity data for this assay | |
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