| Assay Method Information | |
| | NR2B Radioligand Binding Assay |
| Description: | This example describes an NR2B receptor binding assay in rat brain using [3H] (E)-N1-(2-methoxybenzyl)-cinnamidine (see below). The binding assay method was adapted from a previously described cellular human NR1a/NR2B cloned receptor assay (Kiss et al. Neurochem. Internatl. 2005, 46:453-464) to rat brain tissue. This assay serves as a selective measure of NR2B receptor binding activity in native rat brain receptors. Briefly, the brains of male Wistar rats were homogenized (Polytron) before centrifugation at 40,000×g for 15 minutes at 4° C. After 2 washes, the final pellet was homogenized and stored at −80° C. The protein concentration was determined by Bradford assay. [3H] (E)-N1-(2-methoxybenzyl)-cinnamidine was used at 2 different concentrations, 0.5 and 30 nM, with 30 μg of membrane proteins. Non-specific binding was assessed in the presence of excess (10 μM) (E)-N1-(2-nethoxybenzyl)-cinnamidine. A high affinity site was identified, for which inhibition constant (Ki) values of 0.193, 0.176, 0.41, and 0.087 (mean=0.22) nM were determined in independent assays for (E)-N1-(2-methoxybenzyl)-cinnamidine. In cloned human NR1a/NR2B receptors, the Ki values for (E)-N1-(2-methoxybenzyl)-cinnamidine were 1.0 nM and 0.7 nM using [3H] (E)-N1-(2-methoxybenzyl)-cinnamidine and [3H]-ifenprodil as the radioligands, respectively (Claiborne et al. Bioorg. Med. Chem. Lett. 2003, 13:697-700). |
| Affinity data for this assay | |
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