| Assay Method Information | |
| | Inhibition of Specific Binding to the Rat NR1/NR2B Receptor |
| Description: | Male Wistar rats (180 to 200 g) were killed by suffocation in a CO2 chamber for two minutes. Whole brains without cerebellum were removed and dissected on ice, placed into closed vials and stored at −70° C.Membrane fractions were prepared and tested using standard techniques. At the time of the assay, 1 g of the brains was placed into 25 ml of 50 mM Tris/10 mM EDTA buffer, pH 7.1, (25 vol. per g of original tissue) and homogenized for 30 sec at 20000 rpm with an Ultraturrax T25 (Jahnke & Kunkel, IKA-Labortechnik, Staufen, Germany). The homogenate was centrifuged at 4° C. for 10 min at 48000 g (OPTIMA L-70, Beckman, Palo Alto, Calif. 94304, USA).The supernatant was discarded and the pellet was homogenized on ice for 30 sec at 20000 rpm with an Ultraturrax and again centrifuged at 48000 g for 30 minutes at 4° C. The resulted pellet was resuspended in 25 ml of 50 mM Tris/10 mM EDTA buffer, homogenized for 30 sec with an Ultraturrax, aliquoted, frozen at −70° C. and stored until use.After thawing on the day of the assay, a 5 ml membrane aliquot was centrifuged at 48000 g for 30 min at 4° C. The pellet was resuspended in 5 ml of 5 mM Tris/1 mM EDTA buffer, pH 7.4, homogenized for 30 sec at 20000 rpm with an Ultraturrax and centrifuged at 48000 g for 30 min at 4° C. This was repeated twice. The final pellet was homogenized in 5 ml of 5 mM Tris/1 mM EDTA buffer at 4° C. with an Ultraturrax and used for the Ifenprodil-binding assay as described in the following.The incubation mixture of 200 μl contained 5 nmol/l [3H]-Ifenprodil, an optimised amount of membrane preparation, 5 mM Tris/1 mM EDTA (pH 7.4, 10 μM R(+)-3-PPP, 1 μM GBR-12909, 1 μM GBR-12935) and test compound in 1% DMSO. Nonspecific binding was estimated in the presence of 10M CP101.606. The samples were incubated for 60 min. at 4° C.The incubation was terminated by filtration of the membrane preparations using Filtermat B (Pharmacia, Uppsala Sweden) and a Micro Cell Harvester (Skatron, Lier, Norway). The Filtermat B had been presoaked with 1% polyethylene imine and carefully washed with 50 mM Tris/HCl-buffer pH 7.7 after the filtration to separate free and bound radioactivity. The filters were counted in a scintillation counter (Betaplate 1205, Berthold, Wildbad, Germany) in order to determine the specific binding of [3H]-Ifenprodil.The optimal amount of membrane preparation in the assay had been determined and optimized for each membrane preparation separately before the test.Test compounds were either screened at 6 to 10 increasing concentrations for the determination of IC50 and Ki or at 2-4 concentrations for the determination of the percent inhibition. For pipetting of the incubation mixture we routinely used the robot Biomek2000 (Fa. Beckman).For determination of IC50 values the Hill-plot, 2-parameter-model was used. In the NR1/NR2B binding assay a dissociation constant (KD) of [3H]-Ifenprodil of 9 nM was determined. |
| Affinity data for this assay | |
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