| Assay Method Information | |
| | ENPP1 Enzyme Assay with eGAMP Substrate |
| Description: | ENPP1 hydrolyzes nucleotides or nucleotide derivatives to produce nucleoside -5′-monophosphate and pyrophosphate. in addition, ENPP1 hydrolyzes 2′,3′-cGAMP to produce 5′-adenosine monophosphate (AMP) and 5′-guanosine monophosphate (GMP). AMP produced from the reaction is measured using the AMP-Glo kit (Promega). The AMP-Glo kit contains two reagents. The first reagent terminates the AMP-producing enzymatic reaction, removes ATP and converts the produced AMP into ADP. The second reagent converts ADP to ATP which is used to generate luminescence in the luciferase reaction. The amount of luminescence measured is proportional to the amount of AMP produced by ENPP1.[1071]The final reaction mixture contains 50 mM Tris (pH 8.5) buffer, 250 mM NaCl, 0.5 mM. CaCl2, 1 μM ZnCl2, 5% glycerol and 1% DMSO. Serially diluted ENPP1 inhibitors (typically in the range of 10 μM to 0.5 nM) were pre-stored with human recombinant ENPP1 enzymes (R & D systems) at 3 ng/reaction and room temperature (RT) for 5 to 10 minutes. The reaction was initiated by addition of cGAMP (at a final concentration of 5 μM) and reacted at 37 C. for 90 minutes. At the end of the reaction, 10 μl of the AMP-Glo first reagent was added to stop the reaction and the result was stored at room temperature for hour. After storage, 20 μl of an AMP detection solution (a mixture of AMP-Glo II reagent and Kinase-Gio at a ratio of 1:100) was added and stored at room. temperature for 1 hour. Luminescence signals were measured using a Victor plate reader (Perkin Elmer). The maximal activity control (containing enzymes and substrates only in the presence of 1% DMSO; MAX) and the minimal activity control (containing substrates and 1% DMSO; MIN) were evaluated simultaneously. In each experiment, serially diluted reference ENPP1 inhibitors were tested together. IC50, indicating % residual activity versus compound concentration was determined by fitting inhibition curves using the 3-parameter analysis of GraphPad Prism software. Serially diluted samples of one compound were tested two or more times and an average IC50 of each compound was calculated. |
| Affinity data for this assay | |
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