| Assay Method Information | |
| | [125I]-7OH-PIPAT Binding Assay |
| Description: | [125I]-7OH-PIPAT Binding Assay at rat native D3 receptor on membranes from rat ventral striatum. Homogenates from frozen rat brain ventral striatum (nucleus accumbens and olfactory tubercles), were prepared as described by Burris et al. (1994). [125I]-7OH-PIPAT binding assay at D3 receptors was performed in 50 mM Tris-HCl (pH 7.0), 50 mM NaCl, 100 μM Gpp(NH)p (Guanosine 5′-[β,γ-imido]triphosphate) and 0.02% BSA, i.e. conditions which inhibit the [125I]-7-OH-PIPAT binding to D2 and 5HT1A receptors. Compounds of invention were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final in the assay). Displacement experiments were performed in the presence of 0.2 nM [125I]-7OH-PIPAT. The reaction, carried out in a final volume of 200 μl, was initiated by the addition of membrane suspension (about 20 μg/well protein) and lasted 45 min at 37° C. Non specific binding (NSB) was determined in the presence of 1 μM SB277011A. The binding reaction was stopped by rapid filtration through GF/C filterplates pre-soaked in 0.5% polyetylenimmine (PEI) using a Packard cell harvester. After washing with ice-cold 50 mM Tris (pH 7.4) and addition of Microscint 20 (50 μl/well, PerkinElmer), radioactivity was counted with a TopcCount (PerkinElmer). Data were analyzed by non-linear regression analysis using GraphPad Prism 5.0 (GraphPad Software). Ref: Burris, K. D.; Filtz, T. M; Chumpradit, S.; Kung, M. P.; Foulon, C.; Hensler, J. G.; Kung, H. F.; Molinoff P. B. Characterization of [125I](R)-trans-7-hydroxy-2-[N-propyl-N-(3′-iodo-2′-propenyl)amino]tetralin binding to dopamine D3 receptors in rat olfactory tubercle. J. Pharmacol. Exp. Ther. 1994, 268, 935-942. |
| Affinity data for this assay | |
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