| Assay Method Information | |
| | Vanin-1 Recombinant Enzyme Activity Inhibition Assay |
| Description: | A certain mass of the compound was weighed precisely, and prepared with DMSO and reaction buffer (50 mM Tris base, 50 mM KCl, 1.6 mM cysteamine, 0.005% Brij 35, pH 8.0, prepared when using) to a maximum concentration of 10000 nM, then diluted in a 4-fold gradient, and prepared into 10 compound working solutions with different concentrations;for the activity inhibition reaction of recombinant human Vanin-1 (Biorab, JN0618), 2.5 μL of compound working solution and 5 μL of recombinant human Vanin-1 protein were first mixed. The mixture was incubated at room temperature for 15 minutes, then added with 2.5 μL of Pantetheine 7-amino-4-trifluoromethylcoumarin substrate, such that the final concentration of recombinant human Vanin-1 was 62.5 pM and the final concentration of Pantetheine 7-amino-4-trifluoromethylcoumarin substrate was 45 μM in the 10 L reaction system. The reaction was carried out in a 384-well plate (PerkinElmer, 6007280) with DMSO at a final concentration of 1%. The excitation light was set at 405 nm and the emission light was set at 505 nm on the microplate reader, and kinetic reading was performed at 25° C. for 1 hour. |
| Affinity data for this assay | |
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