| Assay Method Information | |
| | PARP2 Enzymatic Activity Assay |
| Description: | 1) 100 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 the next step of blocking. 3) 50 μL of blocking buffer was transferred to the 384-well reaction plate, which was allowed to stand for 1 hour. 4) After the blocking was completed, the blocking buffer (1×PBS, 0.05% Tween-20, 5% BSA) was discarded. The reaction plate was washed three times with PBST solution according to step 2 and finally patted dry. 5) 25/10×PARP2 solution was prepared. 10 μL of PARP2 solution was transferred to a 384-well reaction plate, and 10 μL of reaction buffer (50 mM HEPES (pH 7.5), 0.002% Tween-20, 0.1% BSA, 100 mM NaCl, 2 mM DTT) was transferred to the negative control wells with a final PARP2 concentration of 1.5 nM. 6) 2000× compound was prepared. 50 μL of compound was transferred with echo, then added with 19.95 μL of reaction buffer, and mixed well. 5 μL of mixed compound was transferred to a 384-well reaction plate. 7) 25/10× Biotin-NAD+ solution was prepared. 10 μL of NAD+ solution was transferred to a 384-well reaction plate with a final NAD+concentration of 2 μM, and the plate was incubated at room temperature for 60 minutes. 8) After the reaction was completed, the reaction buffer was discarded. The reaction plate was washed three times with PBST solution according to step 2. 9) Stre-HRP solution was prepared with dilution of blocking buffer. 25 μL of Stre-HRP solution was transferred to the reaction plate with a final Stre-RP concentration of 0.1 μg/mL, and the plate was incubated at room temperature for 1 hour. 10) The Stre-HRP solution was discarded. The reaction plate was washed three times with PBST solution according to step 2. 11) Femto-ECL Substrate A, Femto-ECL Substrate B, and QuantaRed ADHP were mixed at a ratio of 50:50:1, and 25 μL of the mixture was transferred to the 384-well reaction plate. The plate was incubated at room temperature for 10 minutes, and 2.5 μL of QuantaRed stop solution was added thereto. The fluorescence value (Ex 550/Em 620) was read using Paradigm. |
| Affinity data for this assay | |
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