| Assay Method Information | |
| | ENPP1 Activity Assay (32P cGAMP)) |
| Description: | Radiolabeled 32P cGAMP was synthesized by incubating unlabeled ATP (1 mM) and GTP (1 mM) doped with 32P-ATP with 2 μM purified recombinant porcine cGAS in 20 mM Tris pH 7.5, 2 mM MgCl2, 100 μg/mL herring testes DNA overnight at room temperature, and the remaining nucleotide starting materials were degraded with alkaline phosphatase for 4 h at 37° C. The probe 32P-cGAMP (5 μM) was incubated with purified recombinant mouse ENPP1 (20 nM) in 100 mM Tris, 150 mM NaCl, 2 mM CaCl2, 200 μM ZnCl2, pH 7.5 at room temperature for 5 hours. To generate enzyme inhibition curves, 5-fold dilutions of ENPP1 inhibitor were included in the reaction. Degradation was evaluated by TLC (as described by Li et al. Nat. Chem. Biol. (2014) 10:1043-8). Plates were exposed on a phosphor screen (Molecular Dynamics) and imaged on a Typhoon 9400 and the 32P signal was quantified using ImageJ. Inhibition curves were fit to obtain IC50 values using Graphpad Prism 7.03. |
| Affinity data for this assay | |
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