| Assay Method Information | |
| | Determination of the Inhibitory Activity of the Compounds of the Present Invention on PARP1 Enzyme |
| Description: | 1. Experimental ObjectiveThe experimental objective of this Test Example is to determine the inhibitory activity of the compounds on PARP1 enzyme.2. Experimental InstrumentsCentrifuge (Eppendorf 5810R)Microplate reader (BioTek Synergy H1 or PerkinElmer Envision)Pipette (Eppendorf or Rainin)3. Experimental ReagentsPARP1 Chemiluminescent Assay Kit, purchased from BPS bioscience, article number: 8056920×PBST, purchased from Thermo, article number: 28352PBS, purchased from Gibco, article number: 100100234. Experimental MethodThe inhibitory activity of the compounds on PARP1 enzyme was determined by the chemiluminescence method in the experiment. The experiment was carried out in a 384-well plate. Coating histones in a 384-well plate: 5×histone solution was diluted 5 folds with PBS, added to a 384-well ELISA plate (25 μL per well), and incubated at 4° C. overnight. The coated ELISA plate was rinsed with 1×PBST buffer, blocked with the blocking buffer (Blocking buffer 3 in the kit, 100 μL per well) for 30 to 120 minutes, and rinsed with 1×PBST buffer 3 to 6 times. A mixture of PARP reaction biotin-labeled substrate, activated DNA, 10×PARP buffer and water was added (12.5 μL per well). Compound solutions with different concentrations were formulated using the experimental buffer (10% DMSO aqueous solution containing 1.25 mM DTT). The final detection concentration started from 100 nM, with 3-fold dilution and 8 concentrations. The compound solutions were added to the reaction wells of the 384-well plate (2.5 μL per well). 2.5 μL of 10% DMSO aqueous solution containing 1.25 mM DTT was added to the positive control well and blank well (2.5 μL per well). 10 μL of PARP1 enzyme solution formulated with 1×PARP buffer was added to start the reaction. The plate was centrifuged at 1000 rpm for 1 minute, and reacted at room temperature for 60 minutes. After completion of the reaction, the reaction solution was poured off, and the plate was rinsed with 1×PBST buffer. Streptavidin-HRP solution diluted 50 folds with Blocking buffer 3 was added (25 μL per well), and the plate was incubated at room temperature for 30 minutes. The reaction solution was poured off, and the plate was rinsed with 1×PBST buffer 3 to 6 times. The luminescence reaction solution obtained by mixing ECL substrate A and ELISA ECL substrate B (1:1) was added (50 μL per well) for reaction. The chemiluminescence values were measured immediately with a BioTek Synergy H1 or Envision instrument. |
| Affinity data for this assay | |
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