| Assay Method Information | |
| | ENPP1 Inhibition Assay |
| Description: | Assay was performed in a 30 μL final volume in a white 96-well Half Area plate (Costar,Cat #3693). Dose response curve for the compounds were prepared in duplicates with starting conc. at 5 μM and a three-fold titration was performed. The reaction buffer consisted of 50 mM Tris (pH 8.0), 0.5 mM CaCl2), 1 μM ZnCl2, 250 mM NaCl and 0.1 mg/ml BSA. To the compound dilution, 15 μL of ENPP1 enzyme (R&D Systems; Cat #6136-EN-010) was added to each well (final conc. 1 nM) and the mix was pre-incubated for 15 min at RT. An equal volume (15 μL) containing cGAMP (Sigma, Cat #SML-1229) (final conc. 20 μM) was added to initiate the enzyme reaction and the reaction was incubated for 30 min at RT. The reaction was then stopped by heating at 90° C. for 3 minutes. 10 μl of reaction was then transferred to 384 well white, medium binding plates (Grenier, Cat #781075) to which 10 μl of AMP-Glo Reagent 1 was added and mixed well and incubated for 1 hour at 25° C. Following 60 min incubation, 20 μL of detection mixture was added to the enzyme reaction bringing the final volume to 40 μL. The reaction was further incubated for 60 min at RT after which the plates were read using BioTek Plate reader. |
| Affinity data for this assay | |
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