| Assay Method Information | |
| | POLQ Enzyme Activity Inhibition Assay |
| Description: | Brief introduction to the experimental principle: The N-terminal active peptide segment (MI-N899) of POLQ with ATPase activity was co-incubated with compounds, and then reacted with substrate dT50 under the action of ATP to generate ADP, which participated in the subsequent NADH oxidative coupling enzymatic reaction and catalysed the NADH reaction to generate NAD+. The Envision microplate reader from Perkin Elmer Inc was used to measure the reduction of OD value of NADH at 340 nm, thus reflecting the enzyme activity.Experimental instruments: Echo 650 pipetting system from Labcyte Inc, Envision microplate reader from Perkin Elmer Inc; 5810R centrifuge from Eppendorf Inc, and BSD-YX3400 thermostatic shaker from Boxun Inc.Experimental MaterialsReagent BrandPOLQ (N) Synthesized by Pharmaron IncATP SigmadT50 Synthesized by Genscript IncNADH RochePEP SigmaLactate dehydrogenase SigmaPyruvate kinase Sigma384-well plate Greiner Bio-OneExperimental method: The POLQ enzyme was diluted to 100 nM with a reaction buffer (20 mM Tris HCl (pH 7.80), 80 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.01% w/v bovine serum albumin, 0.01% v/v Tween-20, and 5% v/v glycerol). The compounds to be tested were diluted to different concentrations in dimethyl sulfoxide (DMSO) using the Echo 650 pipetting system, and transferred to a 384-well plate, 20 μL/well of 100 nM POLQ was added, and the mixture was incubated at room temperature for 15 minutes. A reaction mixture solution was formulated, and the concentration of each component in the reaction mixture solution was: 100 μM ATP, 300 nM dT50, 300 μM NADH, 6 mM PEP, 10 U/mL lactate dehydrogenase and 20 U/mL pyruvate kinase. 20 μL/well of reaction mixture solution was added to start the enzyme reaction. The final concentration of the compounds in the reaction system started from 10 μM, and was subjected to 3-fold gradient dilution. The concentration range was from 10 μM to 0.0005 μM, and the final concentration of DMSO in the system was 0.2% v/v. The 384-well plate was reacted at room temperature for 20 minutes, and then the OD value at 340 nm was read with an Envision microplate reader. |
| Affinity data for this assay | |
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