| Assay Method Information | |
| | Methods for Determining VMAT2 Inhibitory Activity of a Compound |
| Description: | The human VMAT2 Ki values for the compounds listed in Table 15A and Table 15B were determined using the following procedures. Compound dilution series in DMSO were generated from either powder stocks by hand, or from DMSO stocks using serial dilution on a Vantage (Hamilton), or direct dilution using an Echo 655 (Beckman). In a total volume of 0.145 to 0.150 mL in low-binding 96-well plates (Corning #3605), twelve concentrations of test compound were competed against 10 nM 3H-dihydrotetrabenezine (American Radiolabeled Chemicals) on human platelet homogenate (30 μg membrane protein per well) in VMAT2 binding buffer (Dulbecco's phosphate buffered saline, 1 mM EDTA, pH 7.4). Following incubation at 25° C. for 90 minutes, bound radioligand was collected by rapid filtration onto either GF/B or GF/C glass fiber filters, pretreated with 0.100 polyethylenimine, using either a Unifilter-96 Harvester (PerkinElmer) or Microlab Star (Hamilton). Following harvesting the filter plates were washed with 0.8 mL VMAT2 binding buffer, and bound radioligand was quantified by scintillation counting using a Topcount NXT or Microplate Counter Microbeta (PerkinElmer). |
| Affinity data for this assay | |
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