| Assay Method Information | |
| | [3H]Dopamine ([3H]DA) Uptake Assay |
| Description: | Inhibition of [3H]DA uptake was conducted using isolated synaptic vesicle preparations (Teng et al., 1997). Briefly, rat striata were homogenized with 10 up-and-down strokes of a Teflon pestle homogenizer (clearance 0.003) in 14 ml of 0.32 M sucrose solution. Homogenates were centrifuged (2,000 g for 10 min at 4 C.), and then the supernatants were centrifuged (10,000 g for 30 min at 4 C.). Pellets were resuspended in 2 ml of 0.32 M sucrose solution and subjected to osmotic shock by adding 7 ml of ice-cold MilliQ water to the preparation. After 1 min, osmolarity was restored by adding 900 ul of 0.25 M HEPES buffer and 900 ul of 1.0 M potassium tartrate solution. Samples were centrifuged (20,000 g for 20 min at 4 C.), and the supernatants were centrifuged (55,000 g for 1 hr at 4 C.), followed by addition of 100 ul of 10 mM MgSO4, 100 ul of 0.25 M HEPES and 100 ul of 1.0 M potassium tartrate solution prior to the final centrifugation (100,000 g for 45 min at 4 C.). Final pellets were resuspended in 2.4 ml of assay buffer (25 mM HEPES, 100 mM potassium tartrate, 50 uM EGTA, 100 uM EDTA, 1.7 mM ascorbic acid, 2 mM ATP-Mg2+. pH 7.4). Aliquots of the vesicular suspension (100 ul) were added to tubes containing assay buffer, various concentrations of compound (0.1 nM-10 mM) and 0.1 uM [3H]DA in a final volume of 500 ul, and incubated at 37 C. for 8 min. Nonspecific uptake was determined in the presence of the standard compound, Ro4-1284 (10 uM). Reactions were terminated by filtration, and radioactivity retained by the filters was determined by liquid scintillation spectrometry (Tri-Carb 2100TR liquid scintillation analyzer; PerkinElmer Life and Analytical Sciences, Boston, MA). |
| Affinity data for this assay | |
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