| Assay Method Information | |
| | Determination of Affinity of Human D2L Receptors in Competitive Binding Assay Using [3H]Spiperone |
| Description: | Table 4: CHO-K1 cells expressing human recombinant D2L receptors were washed with phosphate-buffered saline (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 phenylmethylsulfonyl fluoride (PMSF). The resulting 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 then aliquoted and stored at −70° C.Aliquoted membrane preparations were incubated with 0.16 nM [3H]spiperone (PerkinElmer) in the presence or absence of test compound in 96-well plates for 120 minutes at 25° C. in an incubation buffer containing 50 mM Tris-HCl, 1.4 mM ascorbic acid, 0.001% BSA, 150 mM NaCl, pH 7.4, with 1% DMSO in a final reaction volume of 222 μL. Non-specific binding (NSB) was determined in the presence of 10 μM haloperidol (Sigma-Aldrich). After incubation, samples were filtered over UniFilter® GF/C plates (PerkinElmer), washed and Microscint™-20 scintillation cocktail was added. Radioactivity was determined with a MicroBeta2® microplate counter (PerkinElmer).From 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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