| Assay Method Information | |
| | ENPP1 Enzyme Activity Assay |
| Description: | 3 nM mouse ENPP1 was incubated with 5 uM cGAMP and 5-fold serial dilutions of compounds in buffer containing 50 mM Tris pH 7.6, 250 nM NaCl, 500 uM CaCl2, and 1 uM ZnCl2 (total reaction volume=10 μL) at room temperature for 3 hours, after which the reactions were heat inactivated at 95° C. for 10 minutes. The AMP degradation product was converted to ATP, which was detected using luciferase. To achieve this, an enzyme mixture of polyphosphate:AMP phosphotransferase (PAP) and myokinase was prepared according to Goueli et al. in EP2771480. Briefly, PAP was diluted to 2 mg/mL in buffer containing 50 mM Tris pH 7.5, 0.1% NP-40. Myokinase was diluted to 2 KU/mL in buffer containing 3.2 mM ammonium sulfate pH 6.0, 1 mM EDTA, and 4 mM polyphosphate. The heat-inactivated ENPP1 reaction was incubated with PAP (0.01 μg/μL) and myokinase (0.0075 U/μL) in buffer containing 40 mM Tris pH 7.5, 0.05 mg/mL Prionex, 5 mM MgCl2, 20 μM polyphosphate, and 0.15 g/L phenol red (for ease of pipetting) for 3 hours (total reaction volume=20 μL). CellTiterGlo (20 uL) was added to the reaction according to manufacturer's protocol and luminescence was measured. Data were normalized to 100% enzyme activity (no compound) and 0% enzyme activity (no enzyme) before being fit to the function 100/(1+([compound]/IC50)). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |