Radioligand Binding Assays
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For all filtration radioligand binding assays, membrane preparations expressing the target receptors or receptor combinations are resuspended in radioligand binding buffer (15 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 1.25 mM CaCl2, and 1.25 mM MgCl2, pH 7.4), and the membrane suspension is mixed with the appropriate concentrations of radioligand and nonlabeled drugs in 96-well plates in a total volume of 200 μL and incubated for 60 min at the appropriate temperature. At the end of the incubation, membranes are filtered onto Whatman Unifilter preincubated with 0.1% polyethyleneimine in wash buffer (50 mM Tris-HCl, pH 7.4) with a Filtermate 196 harvester and washed three times with ice-cold wash buffer. Radioactivity captured on the filter is quantified on a Topcount microplate scintillation counter with quenching correction after the addition of 45 μL of MicroScint 40 per well and shaking for 20 min. The concentration of membranes and incubation time is determined for each assay in pilot experiments.
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References:
1. Lindemann, L., Jaeschke, G., Michalon, A., Vieira, E., Honer, M., Spooren, W., Porter, R., Hartung, T., Kolczewski, S., Buttelmann, B., Flament, C., Diener, C., Fischer, C., Gatti, S., Prinssen, E. P., Parrott, N., Hoffmann, G. and Wettstein, J. G. (2011) CTEP: a novel, potent, long-acting, and orally bioavailable metabotropic glutamate receptor 5 inhibitor. J Pharmacol Exp Ther. 339, 474-486
2. Michalon, A., Sidorov, M., Ballard, T. M., Ozmen, L., Spooren, W., Wettstein, J. G., Jaeschke, G., Bear, M. F. and Lindemann, L. (2012) Chronic pharmacological mGlu5 inhibition corrects fragile X in adult mice. Neuron. 74, 49-56
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