| Assay Method Information | |
| | RBC HotSpot Poly(ADP-ribose) Polymerases 1 (PARP1) and Poly(ADP-ribose) Polymerases 2 (PARP2) Assays |
| Description: | i. PARP1 assay: Human recombinant PARP1 at a final concentration of 2.5 nM was combined with histone H4 (20 μM) and test articles at various concentrations in reaction buffer (50 mM Tris-HCL, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% DMSO, and 20 μg/mL activated DNA). The solution was inoculated for 20 min at room temperature and the reaction initiated by adding [adenylate-32P]-Nicotinamide Adenine Dinucleotide, 32P-NAD+ at a final concentration of 10 μM. After incubation for 2 hrs at room temperature, the reaction mixture was filtered and washed with 0.75% phosphoric acid for radioactivity detection.ii. PARP2 assay: Human recombinant PARP2 at a final concentration of 2.5 nM was combined with histone H3 (20 μM) and test articles at various concentrations in reaction buffer (50 mM Tris-HCL, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% DMSO, and 20 μg/mL activated DNA). The solution was inoculated for 20 min at room temperature and the reaction was initiated by adding [adenylate-32P]-Nicotinamide Adenine Dinucleotide, 32P-NAD+ at a final concentration of 10 μM. After incubation for 2 hrs at room temperature, the reaction mixture was filtered and washed with 0.75% phosphoric acid for radioactivity detection. |
| Affinity data for this assay | |
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