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Assay Method Information

Assay Name:  Inhibition Assay
Description:  Two-electrode voltage-clamp recordings were made from Xenopus laevis oocytes expressing recombinant rat GluN1/GluN2A, GluN1/GluN2B, GluN1/GluN2C, GluN1/GluN2D, GluA1, or GluK2 receptors following injection of 5-10 ng of cRNA synthesized according to manufacturers' protocols (Ambin, mMessage, mMachine). cDNAs used were rat GluN1-1a (GenBank accession numbers U11418 and U08261; hereafter GluN1), GluN2A (D13211), GluN2B (U11419), GluN2C (M91563), GluN2D (L31611), GluA1 (X17184), GluK2 (Z11548). The current under voltage-clamp was recorded during perfusion with recording solution containing (in mM) 90 NaCl, 1.0 KCl, 0.5 BaCl2, 0.005 EDTA, and 10 HEPES at pH 7.4 (23 C.). Glass micropipettes had resistances of 0.3-1.0 MS2 and were filled with 3.0 M KCl. The membrane potential was clamped at −40 mV during the experiment. Recordings were digitized at 10 Hz and analyzed off line. 20 mM stock solutions of test compounds in 100% DMSO were made and diluted to obtain the final concentration; final DMSO content was 0.05-0.5% (vol/vol). Oocytes expressing GluK2 homomeric receptors were first treated with 10 μM concanavalin A (10 minutes). NMDA receptor responses were obtained by challenging oocytes with 100 μM glutamate plus 30 μM glycine; GluA1 and GluK2 receptors responses were recorded during application of 100 μM glutamate. We recorded the response to 5-7 concentrations of test drug co-applied with glutamate and glycine in 5 or more oocytes obtained from two different frogs.
Affinity data for this assay
 

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Last update November 1, 2007
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