| Assay Method Information | |
| | Determination of Affinity at Human D3 Receptors in Competitive Binding Assay Using [3H]spiperone |
| Description: | Table 8: CHO-K1 cells expressing human recombinant D3 receptors were washed with PBS. Cells were scraped from the plates and centrifuged at 1000×g. Cells were disrupted using a Teflon® pestle homogenizer in buffer containing 25 mM Tris-HCl, pH=7.4, 6 mM MgCl2, 1 mM EDTA, 10 mM PMSF. The suspension was centrifuged at 1000×g. The supernatant was collected and centrifuged at 41,000×g. The supernatant was discarded, and the pellet was resuspended in above buffer. The membrane preparation was aliquoted and stored at −70° C.Aliquoted membrane preparations were incubated with 0.7 nM [3H]spiperone (PerkinElmer) in the presence or absence of test compound in 96-well plates for 120 minutes at 37° C. in an incubation buffer containing 50 mM Tris-HCl, 1.4 mM ascorbic acid, 0.001% bovine serum albumin, and 150 mM NaCl, at pH 7.4, with 1% DMSO in a final reaction volume of 222 L. Non-specific binding (NSB) was determined in the presence of 25 μM (S)-(−)-sulpiride (Sigma Aldrich). After incubation, samples were filtered on GF/C filter plates (PerkinElmer), washed, and Microscint™-20 scintillation cocktail was added. Radioactivity was determined in a MicroBeta2® microplate counter (PerkinElmer).From raw scintillation counts, NSB was subtracted to yield specific binding which was normalized to vehicle-treated samples and converted to displacement % values. IC50 values were determined by a non-linear, least squares regression analysis using MathIQ™ (ID Business Solutions Ltd., Surrey, UK). |
| Affinity data for this assay | |
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