CHEMBL3705172

Radio Ligand Binding Assay: Serial dilutions of compounds (stock in 10 mM DMSO) are prepared by first diluting the compounds in DMSO followed by a 1:50 dilution into assay buffer (10 mM HEPES, pH 8.0, 100 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid-free BSA, 0.05% Tween-20). The radioligand [3H]4 (example 39) (specific activity 1500 GBq/mmol) is diluted directly into the assay buffer immediately before use to obtain a 20 nM solution. The desired amount of membranes (20 μg/well) is diluted with assay buffer. 50 μL of pre-diluted compound and 50 μL of [3H]4 (example 39) is placed into the bottom of a 96-well well plate. 100 μL of the membrane-suspension is added and the plate stirred for 60 minutes. The reaction is stopped by transfer onto the filter of a 96-well GF/C filter plate (soaked for 1 hour in 0.25% PEI) using a cell harvester.