| Assay Method Information | |
| | Inhibition of Compounds on PARP1/7 Enzyme Activity |
| Description: | (1) Pre-coating: Add 100 μL of a PBS buffer (10 mM NaH2PO4, 10 mM Na2HPO4, 150 mM NaCl, pH 7.4) containing 20 g/mL of histone to each well of a 96-well plate, and incubate at 4° C. overnight.(2) Add 30 μL of reaction buffer (50 mM Tris, 2 mM MgCl2, pH 8.0) containing 100 μM NAD+, 25 μM biotinylated NAD+, and 200 nM slDNA to each well.(3) Add 5 μL of the test substance or solvent to each well.(4) Add 20 μL of(PARP1 or PARP7 (50 ng/well), and incubate at 30° C. for 1 hour.(5) Add 50 μL of streptavidin-HRP to the reaction mixture, and incubate at 30° C. for 30 minutes.(6) Finally, add 100 μL of a citrate buffer containing H2O2 and luminol (0.1 M, pH 5.4), and measure the luminescent signal using a microplate reader (Molecular Devices SpectraMax M5).(7) Calculate the inhibition rate of PARP1 or PARP7 enzyme activity as [(control group−treatment group)/control group]×10000. Fit the dose-response data with standard dilutions using Prism GraphPad software and calculate the concentration required to achieve 5000 inhibition of PARP1 or PARP7 enzyme activity (IC50). |
| Affinity data for this assay | |
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