| Assay Method Information | |
| | In Vitro Enzymology Experiment of PARP2, 3, 5A, 5B, 7, 12, and 15 |
| Description: | 1. Preparation of 384-well histone-coated plate: 25 μL of histone solution was added to each well, and the plate was incubated overnight at 4° C.2. Preparation of PBST buffer, blocking buffer, and assay buffer3. The 384-well histone-coated plate was washed three times with PBST buffer. The plate was blocked by 50 μL of blocking buffer for 1 hour at room temperature. The plate was then washed three times with PBST buffer.4. Compound preparation:2000× compound was prepared in a 96-well source plate. 50 nL of the compound was transferred from the source plate to a 96-well intermediate plate, and 39.95 μL of assay buffer was added to each well. The plate was shaken well and centrifuged at 1000 rpm for 1 minute.5 μL of compound DMSO solution was transferred to each well.5. Enzymatic reaction:An enzyme mixture was incubated at 25° C. for 10 minutes.10 μL of the enzyme mixture was added, and incubated with the compound at room temperature for 10 minutes.10 μL of assay buffer was added to the negative control well of the assay plate.10 μL of 2.5× Biotin-NAD+ was added to each well, and the plate was incubated at 25° C. for 60 minutes.The plate was then washed three times with PBST buffer.6. Detection:25 μL of Stre-HRP was added.The plate was incubated at room temperature for 1 hour and washed three times with PBS buffer.25 μL of QuantaRed Enhancer mix was added. The plate was incubated for 10 minutes.2.5 μL of QuantaRed Stop Solution was added to terminate the peroxidase reaction, and the plate was shaken for 10 to 30 seconds.7. Paradigm was used to read the plate immediately to detect the readings at Ex550/Em620.8. Data processingUsing equation (1) to fit the data in Excel to obtain inhibition valuesinh%=(Max-Signal)/(Max-Min)*100Equation(1)Using equation (2) to fit the data in XL-Fit to obtain IC50 valuesY=Bottom+(Top-Bottom)/(1+(IC50/X)*HillSlopeEquation(2)Y is the percentage of inhibition, and X is the compound concentration. |
| Affinity data for this assay | |
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