| Assay Method Information | |
| | n Vitro Biochemical Test of Inhibiting SARM1 Enzyme Activity Assay |
| Description: | First, 200 μM of the compound was added to a 50 mM Tris-HCl (pH 7.5) solution containing 0.05 μg/ml dN-SARM1. Then half of the mixture was added to an equal volume of 50 mM Tris-HCl (pH 7.5) solution containing 0.05 μg/ml dN-SARM1 (pH 7.5) for mixing. The drug was diluted 6 times by analogy to final concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125 μM, or 200, 50, 12.5, 3.125, 0.78, 0.195, 0.049 μM. The solution without addition of inhibitor was control group. The above solutions were incubated at room temperature for 10 min. Then 50 μM NAD, 50 μM PC6 as substrate and 50 μM NMN as activator were added to the dN-SARM1 protein incubated with inhibitor for 30 min of reaction at room temperature. The concentration of each component was the final concentration in the reaction system.During the reaction process, PC6 fluorescence spectrum kinetics was detected by a microplate reader at an excitation wavelength and an emission wavelength of 390 nm and 520 nm, respectively. Finally, the activity of the protein was denoted by the reaction rate, and the half-inhibition concentration was calculated. The higher the reaction rate, the stronger the protein activity, and the lower the inhibition efficiency of the compound. |
| Affinity data for this assay | |
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