| Assay Method Information | |
| | Enzyme Assay |
| Description: | In a 96-well opaque black plate, NAMPT (30 nM all concentrations provided as final), PRPP (50 μM) and ATP (2 mM) with or without test compounds (11 concentrations, prepared by three-fold dilutions from final concentration of 30 mM, all in triplicate) were incubated for 20 minutes at 37° C. in TMD buffer (50 mM Tris-HCl, 10 mM MgCl2, 2 mM DTT, pH 7.5). The enzymatic reaction was initiated by the addition of NAM (25 μM) and the plate incubated for 20 minutes at 37° C. 20 μL of 20% acetophenone (in DMSO) and 20 μl of 2 M KOH were added to each well and incubated to ambient temperature of 5 minutes, then 90 μl of 100% formic acid was added to each well and the plate incubated at 37° C. for 20 minutes before reading on a Hides Sense plate reader (Ex/Em=355/460 nm). Data was processed to % control in Excel and IC50 curves fitted using GraphPad Prism. Data is reported from fitting of n=3 separate repeats |
| Affinity data for this assay | |
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