| Assay Method Information | |
| | Enzyme Activity |
| Description: | he assay described below is to determine the activity of the compounds of the present invention for inhibiting the enzyme activity of PARP by using the Trevigen HT F homologous poly (adenosine diphosphate-ribose) polymerase inhibition assay kit (TREVIGEN HT F homogeneous PARP Inhibition Assay Kit, No. 4690-096-K). The assays are based on the NAD+ amount needed to be consumed during the DNA repair process, which is also used in another reaction for catalyzing the substrate without fluorescence activity into molecules with high fluorescence activity. Therefore, the NAD+ level in the reaction system can be learned by measuring the enhancement degree of the fluorescence signal, and then the inhibition degree of the test compound on the enzyme activity of PARP was calculated.The instructions of TREVIGEN HT F homologous poly (adenosine diphosphate-ribose) polymerase inhibition assay kit can be used as reference for the detailed operation of the assays as well as the preparation of the reagents, such as the reaction mixture (reaction mix), cycling reaction mixture (cycling mix), a buffer solution (buffer), and the like.The procedures for the assay are summarized as follows: The tested compounds were dissolved in dimethylsulfoxide and then diluted with 1× buffer to the concentration desired in the experiment. 25 μL of a 200 nM NAD+ solution was added to a 96-well round bottomed plate, followed by the addition of 1 μL of tested compounds solution, and the control of replicate wells were installed. Then 25 pt of the reaction mixture containing DNA, PARP enzyme and reaction buffer was added into each well. After incubating for 30 minutes at room temperature, 50 μL of cycling reaction mixture was added into each well and incubated in the dark at room temperature for 15 to 40 minutes. Then 50 pt of stop solution was added into each well and the fluorescence values of each well were read on an ELISA (Ex544 nm, Em590 nm). The inhibition rate of the test compound on the enzyme activity of PARP could be calculated by the standard curve equation of NAD+. |
| Affinity data for this assay | |
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