| Assay Method Information | |
| | ENPP1 Inhibition Assay |
| Description: | Assay Buffer: 1 mM CaCl2), 0.2 mM ZnCl2, 50 mM Tris, pH 9.0. Substrate: 8 mM Thymidine 5′-monophosphate p-nitrophenol ester sodium salt (Sigma Cat #T4510). Enzyme: 5 ng/L Recombinant Human ENPP-1 Protein (R&D Cat #6136-EN-010) in DMSO in 96-well clear assay plates. 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 and 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+4 μL Enzyme. Both the enzyme and substrate was 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 45 minutes. The reaction was quantified by measuring absorbance at 405 nm using the Envision. IC50 values are determined using GraphPad Prism 5 software. The data were entered as an X-Y plot into the software as percent inhibition for each concentration of the drug. The concentration values of the drug were log transformed and the nonlinear regression was carried out using the sigmoidal dose-response (variable slope) option within the GraphPad software to model the data and calculate IC50 values. The IC50 values reported are the concentration of drug at which 50% inhibition was reached. |
| Affinity data for this assay | |
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