| Assay Method Information | |
| | PARP-1 Enzymatic Assay |
| Description: | Table 4: T Universal Chemiluminescent PARP Assay kit (purchased from TREVIGEN); PBS (phosphate buffer saline, purchased from Wisent); Triton X-100 (purchased from Macklin); Envision Multimode Plate Reader (PerkinElmer). (I) Preparation of Reagents:1. Washing solution: Triton X-100 was added to 1×PBS with a final concentration of Triton X-100 of 0.1%.2. 1×PARP buffer: The 20×PARP buffer in the kit was 20-fold diluted with water to prepare a 1×PARP buffer, which was used to prepare the compounds, enzyme solutions and substrate solutions.3. 1×Strep-Diluent solution: The 10×Strep-diluent in the kit was 10-fold diluted with water to prepare a 1×Strep-diluent solution.(II) Preparation of Test Compounds:The test compounds were diluted from 200 μM to 2.56 nM. The DMSO concentration was 100%. 2 μL of each of the inhibitors at various concentration gradients was added to a compound intermediate plate, and 38 μL of the 1×PARP buffer was then added. The two were mixed well for use, and the DMSO concentration was 5% at this time.(III) Procedures:a) The 1×PARP buffer was added to a test plate at 50 μL per well, and the test plate was incubated at 25° C. for 30 min.b) After the incubation was completed, the liquid in the test plate was discarded, and each of the compounds at various concentration gradients was pipetted from the compound intermediate plate and added into the test plate at 10 μL per well. The samples were in duplicate.c) The enzyme solution (0.5 IU) was added to the test plate at 15 μL per well. The compound and enzyme were co-incubated at 25° C. for 10 min.d) After the incubation was completed, 25 μL of the 1×PARP Cocktail (comprising 2.5 μL of 10×PARP Cocktail, 2.5 μL of 10× Activated DNA and 20 μL of 1×PARP buffer) was added to each well of the test plate. The test plate was incubated at 25° C. for 1 h. The final concentrations of the compounds were from 2 μM to 0.0256 nM, and the DMSO concentration was 1%.e) After the incubation was completed, the test plate was washed twice with 200 μL of washing solution per well and then washed twice with 200 μL of PBS per well.f) The Strep-HRP in the kit was 500-fold diluted with the 1×Strep-Diluent solution. The resulting solution was added to the test plate at 50 μL per well, and the test plate was incubated at 25° C. for 1 h.g) After the incubation was completed, the test plate was washed twice with 200 μL of washing solution per well and then washed twice with 200 μL of PBS per well.PeroxyGlow A and B in the kit were mixed at a ratio of 1:1, and the mixed solution was added to the test plate at 100 μL per well. |
| Affinity data for this assay | |
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