| Assay Method Information | |
| | PARP1 Enzymatic Activity Assay |
| Description: | 1) 50 ng/mL of histone coating buffer was prepared with 1×PBS, and 25 μL of coating buffer was transferred to a 384-well reaction plate, which was coated at 4° C. overnight. 2) After the coating was completed, the coating buffer was discarded. The reaction plate was washed with PBST solution (1×PBS, 0.05% Tween-20) by transferring 50 μL of PBST to the 384-well reaction plate, allowing it stand for 5 minutes, discarding the washing buffer, refilling the washing buffer, repeating the washing three times, and finally patting the plate dry for blocking. 3) 50 μL of blocking buffer (1×PBS, 0.05% Tween-20, 5% BSA) was transferred to a 384-well reaction plate, which was allowed to stand for 1 hour. After washing, the blocking buffer was discarded. The reaction plate was washed three times with PBST solution as described above and finally patted dry. 4) 1000× compound was prepared. 1 μL of compound was transferred to a 96-well plate containing 199 μL of reaction buffer (50 mM Tris-HCl (pH 7.5), 0.005% Tween-20, 0.01% BSA) and mixed well. 5 μL of mixed compound was transferred to a 384-well reaction plate. 5) 25/10×PARP1-DNA solution was prepared with reaction buffer. 10 μL of PARP1-DNA solution was transferred to a 384-well reaction plate, and 10 μL of DNA solution was transferred to the negative control wells with a final PARP1 concentration of 0.02 nM and a final DNA concentration of 0.8 nM. 6) 25/10×NAD+ solution was prepared with reaction buffer. 10 μL of NAD+ solution was transferred to a 384-well reaction plate with a final NAD+ concentration of 3.5 μM, and the plate was incubated at room temperature for 60 minutes. 7) After the reaction was completed, the reaction buffer was discarded. The reaction plate was washed three times with PBST solution according to step 2 and finally patted dry. 8) Primary antibody was 2000-fold diluted with the blocking buffer. 20 μL of primary antibody was added to the reaction plate, which was incubated at room temperature for 1.5 hours. 9) The primary antibody was discarded. The reaction plate was washed three times with PBST solution according to step 2 and finally patted dry. 10) Secondary antibody was 2000-fold diluted with the blocking buffer. 20 μL of secondary antibody was added to the reaction plate, which was incubated at room temperature for 1 hour. 11) The secondary antibody was discarded. The reaction plate was washed three times with PBST solution according to step 2 and finally patted dry. 12) Femto-ECL Substrate A and Femto-ECL Substrate B were mixed at a ratio of 1:1, and 25 μL of the mixture was transferred to the 384-well reaction plate. The chemiluminescence value RLU was read with Envision. |
| Affinity data for this assay | |
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