| Assay Method Information | |
| | Human D2 Binding Assay |
| Description: | The assay was performed as a SPA-based competition-binding in a 50 mM Tris pH 7.4 assay buffer containing 120 mM NaCl, 5 mM KCl, 4 mM MgCl2, 1.5 mM CaCl2, and 1 mM EDTA.1.5 nM 3H-raclopride (Perkin Elmer, NET 975) was mixed with test compound before addition of 20 μg of a homogenised human D2 receptor membrane-preparation and 0.25 mg SPA beads (WGA RPNQ 0001, Amersham) in a total volume of 90 μL. The assay plates were under agitation incubated for 60 minutes at room temperature and subsequently counted in a scintillation counter (TriLux, Wallac). The total binding, which comprised approximately 15% of added radioligand, was defined using assay buffer, whereas the non-specific binding was defined in the presence of 10 μM haloperidol. The non-specific binding constituted approximately 10% of the total binding.Data points were expressed in percent of the specific binding of 3H-raclopride and the IC50 values (concentration causing 50% inhibition of 3H-raclopride specific binding) were determined by non-linear regression analysis using a sigmoidal variable slope curve fitting. The dissociation constant (Ki) was calculated from the Cheng Prusoff equation (Ki=IC50/(1+(L/KD)), where the concentration of free radioligand L is approximated to the concentration of added 3H-raclopride in the assay. The KD of 3H-raclopride was determined to 1.5 nM from two independent saturation assays each performed with triplicate determinations. |
| Affinity data for this assay | |
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