| Assay Method Information | |
| | ENPP2 Assay |
| Description: | Table 3: Assay Buffer: 50 mM Tris, 10 mM CaCl2, 5 mM MgCl2, 0.02% Brij-35 (v/v), pH 8.5Substrate: 20 mM Bis(p-Nitrophenyl) Phosphate Sodium Salt (BPNPP) (Sigma, Catalog #N3002) Enzyme: 0.01 ng/μL Recombinant Human ENPP-2/Autotaxin (rhENPP-2) (R&D Catalog #5255-EN) DMSO (Selleck S8218)>96-well clear assay plates Protocol: An eight point serial dilution of drugs was prepared in 10× in assay buffer with the final assay concentrations starting at 10 μM, 3 μM, 1 μM, 0.3 μM . . . 0 μM. A dilution of DMSO was included as a control. The assay plate was set up as follows with each well in duplicate: 81 μL assay buffer+10 μL ENPP1 inhibitor or DMSO+5 μL Substrate+5 μL Enzyme (0.05 ng). Both the enzyme and substrate were added to opposite sides of the well to ensure that there was no interaction until all wells had both components. The plate was then centrifuged gently for 10 seconds, followed by an incubation at 37° C. for 10 minutes. The reaction was quantified by measuring absorbance at 400 nm using the Envision. PF8380 was used as a positive control for the assay. |
| Affinity data for this assay | |
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