| Assay Method Information | |
| | ENPP3 cGAMP Biochemical Assay |
| Description: | Compounds were added to 384 well Proxiplate (PerkinElmer, Waltham MA) at 100 nl/well from DMSO stocks using Echo (Labcyte, San Jose CA). Recombinant human ENPP3 protein (Acro Biosystems, Newwark DE) was prepared in assay buffer (50 mM Tris pH7.5, 1 mM TCEP pH7.0, 2 mM MgCl2, 2 mM CaCl2), 2 mM ZnCl2 and 0.005% Tween20) and added at 2 μl/well. The plate was sealed, centrifuged at 1000 rpm for 1 minute and incubated at room temperature for 30 minutes before 2 μl substrate 2′3′-cGAMP (R&D Systems, Minneapolis MN) in assay buffer was added to each well to a final concentration of 70.5 nM ENPP3 and 120 μM 2′3′-cGAMP. The plate was sealed and centrifuged at 1000 rpm for 1 minute again. After incubation at 25° C. for 60 minutes, Terbium labelled AMP/GMP antibody and AMP/GMP HiLyte647 Tracer (BellBrook Labs, Fitchburg WI) in detection buffer (50 mM Tris pH7.5, 20 mM EDTA and 0.1% Prionex) were added to the reaction mixture to a final concentration of 475 nM and 3 nM respectively. The assay plate was incubated at room temperature for 120 minutes. TR-FRET signal was detected using Envision (PerkinElmer, Waltham MA). |
| Affinity data for this assay | |
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