| Assay Method Information | |
| | Determination of Affinity at Human D3 Receptors in Competitive Binding Assay Using [3H]raclopride |
| Description: | Table 7: Cell cultures (CHO-K1) expressing recombinant human D3 receptors (DRD3, GenBank ID U32499, purchased from Euroscreen Fast, Brussels, BE) were homogenized in 4× buffer (v/v) solution (15 mM Tris, 2 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, pH 7.4 at 25° C.) with a Dounce tissue grinder and centrifuged at 40,000×g at 4° C. for 25 minutes. The supernatant was removed, and the pellet was resuspended in 4× (v/v) buffer and centrifuged. The process was repeated twice, and the pellet was resuspended in storage buffer (75 mM Tris, 12.5 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose, pH 7.4 at 25° C.) at a volume of 12.5 mL/g original cell weight. The membrane preparation was then aliquoted and stored at −70° C.Compounds were diluted in DMSO and binding buffer (containing 50 mM Tris, 5 mM MgCl2, 5 mM KCl, 1 mM CaCl2), 120 mM NaCl, 1 mM EDTA) and 50 μL of each solution was transferred into a deep-well plate (BRAND) in 5-fold final concentration in 5% DMSO-buffer solution. The aliquoted membrane preparation was thawed and washed once in binding buffer. In the same buffer, 3.3 μg protein/assay was incubated with ca. 2.7 nM [3H]raclopride (PerkinElmer) in the presence or absence of test compound for 120 minutes at 25° C. in a volume of 250 μL in a 96-well deep well plate (BRAND). Non-specific binding (NSB) was determined in the presence of 10 μM haloperidol. DMSO final concentration was 1% (v/v) in all reactions. After incubation, samples were filtered over UniFilter® GF/B plates (PerkinElmer) using a Filtermate™ harvester (PerkinElmer) and washed with 4×1 mL ice-cold binding buffer. The plate was dried at 40° C. for an hour and 40 μL Microscint™-20 scintillation cocktail (PerkinElmer) was added to each well. Radioactivity was determined with a Microbeta2® microplate counter (PerkinElmer). |
| Affinity data for this assay | |
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