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Assay Method Information

Assay Name:  Radioligand Binding Assay (Ki) and Inhibition of Substrate Uptake (EC50/IC50)
Description:  Binding affinity of each compound was measured by assessing the potency of inhibition of binding of radiolabeled RTI-55. Membranes were preincubated with compound before the addition of [125I]RTI-55. The reaction was terminated by filtration through Whatman GF/C filters using a 96-well Tomtech cell harvester (Tomtech, Orange, CT). Scintillation fluid was added to each filter spot and radioactivity remaining on the filter was determined using a Wallace beta-plate reader. IC50 values were converted to Ki values using the Cheng-Prusoff equation. Specific binding was defined as the difference in binding observed in the presence and absence of 5 uM mazindol (HEK-hDAT and -NET) or 5 uM imipramine (HEK-hSERT). EC50/IC50 values were obtained from inhibition of the reuptake of [3H] dopamine for DAT, [3H] serotonin for SERT, or [3H] norepinephrine for NET.
Affinity data for this assay
 

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Last update November 1, 2007
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