| Assay Method Information | |
| | Human DAT assay |
| Description: | Binding to dopamine transporter (DAT) in vitro. Human embryonic kidney (HEK) 293 cells (Invitrogen, Zug, Switzerland) stably transfected with human DAT were cultured. The cells were collected and washed three times with phosphate-buffered saline (PBS). The pellets were frozen at −80° C. The pellets were then resuspended in 400 ml of 20 mM HEPES-NaOH, pH 7.4, that contained 10 mM EDTA at 4° C. After homogenization with a Polytron (Kinematica, Lucerne,Switzerland) at 10000 rotations per minute (rpm) for 15 s, the homogenates were centrifuged at 48000×g for 30 min at 4° C. Aliquots of the membrane stocks were frozen at −80° C. All assays were performed at least three times. The test compounds were diluted in 20 ml of binding buffer (252 mM NaCl, 5.4 mM KCl, 20 mM Na2HPO4, 3.52 mM KH2PO4, pH 7.4) and 10 point dilution curves were made and transferred to 96-well white polystyrene assay plates (Sigma-Aldrich, Buchs, Switzerland). [3H]-WIN35,428 ( 86 Ci /mmol; Perkin-Elmer) was the radioligand for the DAT assay and had a Kd of 12 nM. Fifty microliters of [3H]-WIN35,428 ( 40 nM concentration) was added to each well of the hDAT assay plates, targeting a final [3H]-WIN35428 concentration of 10 nM. Twenty microliters of binding buffer alone in the assay plate defined the total binding, whereas binding in the presence of 10 μM indatraline defined nonspecific binding. Frozen DAT membrane stocks were thawed and resuspended to a concentration of approximately 0.04 mg protein/ml binding buffer (1:1 diluted in H2O) using a polytron tissue homogenizer. The membrane homogenates (40 μg/ml) were then lightly mixed for 5-30 min with polyvinyl toluene (PCT) wheat germ agglutinin-coated scintillation proximity assay (WGASPA; Amersham Biosciences) beads at 7.7 mg beads/ml homogenate. One hundred thirty microliters of the membrane/bead mixture were added to each well of the assay plate that contained radioligand and test compounds (final volume in each well, 200 μl) to start the assay, which was incubated for approximately 2 h at room temperature with agitation. The assay plates were then counted in the PVT SPA counting mode of a Packard Topcount. Fifty microliters of the [3H]-WIN35428 stocks were counted in 5 ml of ReadySafe scintillation cocktail (Beckman Industries) on a Packard 1900CA liquid scintillation counter to determine the total counts added to the respective assays. Non-linear regression was used to fit the data to sigmoid curves and determine IC50 values for binding and uptake. Ki values for binding and uptake were calculated using the following Cheng-Prusoff equation: Ki=IC50/(1+[S]/Km). |
| Affinity data for this assay | |
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