| Assay Method Information | |
| | Fluorescence Polarization (FP) Displacement Assay |
| Description: | All FP measurements were performed at room temperature in PBS buffer containing 0.01% Triton-X 100 and 1% DMSO in black 384-well plates. Measurements were performed on a Tecan F200 Pro plate reader fitted with polarized 485(20) nm emission filters and 535(25) nm emission filters. Initial titration of 20 nM FP probe with varying concentrations of NAMPT protein was performed to estimate a Kd value for the probe ( 750 nM), which defined the NAMPT concentration for all subsequent experiments. Anisotropy data from equilibrium competition binding experiments were fit to a four-parameter dose response equation in GraphPad prism. |
| Affinity data for this assay | |
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