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| | General M6PR Binding Assay |
| Description: | M6PR binding was measured in black 96-well plates using a fluorescence polarization assay. A fluorescent probe consisting of a reference M6Pn ligand linked to Cy5 was synthesized. Test compounds were resuspended in DMSO and 3-fold serial dilutions were made at 100x final concentrations. Binding reactions were conducted in 100 µl final volume in 20 mM HEPES (pH 7.5) 100 mM NaCl 0.015% Tween-201% DMSO with 100 nM M6PR (Domains 1-9, R&D Systems) and 1 nM probe. Fluorescence polarization was measured using λex = 620 nm, λem = 688 nm on an Envision plate reader (Perkin Elmer) after 2 hr incubation time. Dose responses were conducted in duplicate and normalized to the response with DMSO (high) and 1 µM reference compound (low) on each plate. IC50 values were determined by fitting to 4-parameter curves in GraphPad Prism. |
| Affinity data for this assay | |
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