Assay Method Information
Assay Name:  Determination of Affinity of Human D2L Receptors in Competitive Binding Assay using [3H]raclopride
Description:  Table 3: Cultured CHO-K1 cells expressing recombinant human D2L receptors (DRD2L cAMP Hunter™, Gi cell line, Eurofins DiscoverX, Fremont, CA, USA) were homogenized in 4× (v/v) buffer solution (50 mM Tris, 5 mM MgCl2, 1 mM EGTA, pH 7.4 at 25° C.) with a Dounce tissue grinder and centrifuged at 40,000×g at 4° C. for 10 minutes. The supernatant was removed, and the pellet was resuspended in 4× buffer (v/v) and centrifuged. The resulting pellet was resuspended in the above buffer solution at a volume of 12.5 mL/g original weight. The membrane preparation was then aliquoted and stored at −70° C.Test compounds were diluted serially in DMSO and subsequently diluted to 5% DMSO (v/w) in binding buffer (50 mM Tris, 5 mM MgCl2, 5 mM KCl, 1 mM CaCl2), 120 mM NaCl, 1 mM EDTA). A 5× concentrated compound in binding buffer (50 μL) was transferred into 96 well deep-well plates (BRAND, Wertheim, Germany). Aliquoted membrane preparations were thawed and washed once in binding buffer. In the same buffer, 10 μg protein/well was incubated with 2 nM [3H]raclopride (PerkinElmer, Waltham, MA, USA) in the presence or absence of test compound (to determine the binding inhibition of the test compound or the total binding, respectively) for 120 minutes at 25° C. in a volume of 250 μL. Non-specific binding (NSB) was determined in the presence of 10 μM haloperidol, an antagonist of the D2 receptor. After incubation, samples were filtered over UniFilter® GF/B plates (PerkinElmer) using a Filtermate™ harvester (PerkinElmer) and washed four times with 1 mL ice-cold binding buffer. The plates were dried at 40° C. for an hour and 40 μL Microscint™-20 scintillation cocktail (PerkinElmer) was added to each well. The radioactivity was determined using a MicroBeta2® microplate counter (PerkinElmer).
Affinity data for this assay
 
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