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| | Binding Assay |
| Description: | For binding experiments, cDNA encoding human mGlu 5a receptor was transiently transfected into EBNA cells using a procedure described by Schlaeger and Christensen [Cytotechnology 15:1-13 (1998)]. Cell membrane homogenates were stored at -80° C. until the day of assay where upon they were thawed and resuspended and polytronised in 15 mM Tris-HCl, 120 mM NaCl, 100 mM KCl, 25 mM CaCl2, 25 mM MgCl2 binding buffer at pH 7.4 to a final assay concentration of 20 ug protein/well.Saturation isotherms were determined by addition of twelve [3H]MPEP concentrations (0.04-100 nM) to these membranes (in a total volume of 200 ul) for 1 h at 4° C. Competition experiments were performed with a fixed concentration of [3H]MPEP (2 nM) and IC50 values of test compounds evaluated using 11 concentrations (0.3-10,000 nM). Incubations were performed for 1 h at 4° C.At the end of the incubation, membranes were filtered onto unifilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.1% PEI in wash buffer, Packard BioScience, Meriden, Conn.) with a Filtermate 96 harvester (Packard BioScience) and washed 3 times with cold 50 mM Tris-HCl, pH 7.4 buffer. Nonspecific binding was measured in the presence of 10 uM MPEP. The radioactivity on the filter was counted (3 min) on a Packard Top-count microplate scintillation counter with quenching correction after addition of 45 ul of microscint 40 (Canberra Packard S. A., Zurich, Switzerland) and shaking for 20 min. |
| Affinity data for this assay | |
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