| Assay Method Information | |
| | Enzyme Activity Assay |
| Description: | Table 2: PARP2, PARP5A, PARP5B, PARP6, PARP7, PARP14 and PARP15 chemical fluorescence detection kits were purchased from BPS Bioscience. The histone solution in the kit was diluted 5× with 1×PBS, and 25 μL of the diluted histone solution was added to a microwell plate and incubated overnight at 4° C. After the incubation, the plate was washed three times with PBST (0.05% Tween-20). 100 μL of the blocking solution was added to the microwell plate and incubated at 25° C. for 90 minutes. After the incubation, the plate was washed three times with PBST. 2.5 μL of compound 4 diluted in test buffer and 5 μL of substrate mixed solution were added to the microwell plate. 5 μL of the diluted PARP enzyme was added to the microwell plate, and the reaction system was incubated at 25° C. for 60 minutes.After the incubation, the plate was washed three times with PBST. Streptavidin-HRP was diluted 50× with a blocking solution, and 25 μL of the diluent was added to the microwell plate and incubated at 25° C. for 30 minutes. After the incubation, the plate was washed three times with PBST. ELISA ECL substrate A and substrate B were mixed at a ratio of 1:1 (v/v), 25 μL of the mixture was added to the microwell plate, and the chemiluminescence value was read.The inhibition rate was calculated according to formula [(1−(RLUsample-RLUmin)/(RLUmax−RLUmin))×100%], where RLUsample was the readout of the compound well, RLUmax was the readout of the solvent control well, and RLUmin was the readout of the control well without the PARP1 enzyme. |
| Affinity data for this assay | |
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