| Assay Method Information | |
| | ENPP1 Inhibitory Activity Assay |
| Description: | Preparation of 1×reaction solution: 50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.5 mM CaCl2), 1 μM ZnCl2, 0.01% Tween-20, 0.01% BSA.Preparation of compound concentration gradient: the tested compound was dissolved in DMSO (dimethyl sulfoxide) to an initial concentration of 10 μM, diluted at 3 times, with 10 concentrations, single well or multi-well testing was set. The initial concentration in the positive control compound STF-32 test was 1 μM, diluted at 3 times, with 10 concentrations, and multi-well testing was set for each concentration. The compound was diluted by gradient in a 384 well plate to a solution with the final concentration of 1000 times (1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15 nM), and then 5 nL of the solution was transferred to a 384 well reaction plate for testing using Echo550. 5 nL of 100% DMSO was transferred from the smallest well (with only substrate, no enzyme, minimum signal value) and the largest well (with substrate, with enzyme, no inhibitor, maximum signal value).20 nM of 2×hENPP1-ECD-His protein solution was prepared using 1×reaction solution.M of 2×2′3′-cGAMP substrate solution was prepared using 1×reaction solution.2.5 μL of 2×protein solution was added to each compound well and the maximum pore of the reaction plate, and 2.5 μL of 1×reaction solution was added to the smallest well.The reaction solution was centrifuged at 1000 rpm for 1 minute and incubated at room temperature for 15 minutes.2.5 μL 2×2′3′-cGAMP substrate solution was added to each well of the reaction plate, centrifuged at 1000 rpm for 1 minute, and incubated at room temperature for 60 minutes.5 μL of R1 solution (from AMP-Glo™ Kit) was added to each well of the reaction plate, centrifuged at 1000 rpm for 1 minute and incubated at room temperature for 120 minutes.10 μL of R2 solution (from AMP-Glo™ Kit) was added to each well of the reaction plate, centrifuged at 1000 rpm for 1 minute and incubated at room temperature for 30 minutes.A multifunctional enzyme-linked immunosorbent assay reader (2104EnVision) was used to detect and the test results were recorded. |
| Affinity data for this assay | |
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