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Assay Method Information
Assay Name:  GTPgS Binding Assay
Description:  The composition of the assay mixtures [in a final volume of 200 μL in 96-well U-bottom plates (Greiner) was as follows: 50 mM Tris-HCl buffer, pH 7.7, 10 mM MgCl2, 0.2 mM EGTA, 2 mM CaCl2, 100 mM NaCl, 20 M guanosine 5′-diphosphate (Sigma), 0.3 nM [35S]GTPgS (1250 Ci/mmol (PerkinElmer)), and the test compounds at increasing concentrations (from 10 nM up to 10 M), 10 g of rat cortical membranes, and a concentration of 1 M GABA, that has been observed in previous experiments to correspond to the EC25, a concentration that gives 25% of the maximal response of GABA. The samples were incubated at room temperature for 60 minutes on a shaker. The incubation was stopped by rapid vacuum filtration over glass-fiber filter plates (UniFilter-96 well, GF/B membrane plates, PerkinElmer) using a 96-well plate harvester (TOMTEK Harvester). The UniFilter plate was washed five times with ice-cold wash buffer (50 mM Tris-HCl buffer, pH 7.7, 10 mM MgCl2, and 100 mM NaCl. After filtration the plate was dried for 90 minutes at 55° C. The plates were closed on the bottom with black sealing membranes, and liquid scintillation cocktail (35 μL, Betaplate Scint, PerkinElmer) was added to each well. After sealing the top of the plate, an additional incubation step of 90 minutes at room temperature followed before measuring the plate. The amount of membrane-bound [35S]GTPgS was measured using a 96-well plate reader (Microbeta , PerkinElmer). Nonspecific binding was measured in the presence of unlabeled 10 μM of GTPgS (Millipore) and without GABA. Basal binding was measured in the absence of 1 μM GABA, and maximal binding was measured in the presence of GABA using 1 mM GABA concentrations.
Affinity data for this assay
 
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Last update November 1, 2007
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