Na+-dependence of [3H]nisoxetine binding to dDAT was determined using 0.5 μg ml−1 of purified dDAT mixed with 12 nM [3H]nisoxetine (80.4 Ci mmol−1, PerkinElmer), 108 nM nisoxetine, and buffer containing 20 mM Tris-HCl, pH 8.0, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 14 μM lipids (3:1:1, POPC:POPE:POPG)) supplemented with the indicated NaCl and KCl concentrations. The ionic strength was maintained by substituting Na+ and K+ with NMDG+. The indicated conditions were added to a clear-bottom, 96-well plate (Corning) and incubated with agitation at 4 °C. 5% (v/v) YSi-Cu His-Tag SPA beads (Perkin Elmer) was added to each well. Plates were sealed, mixed at RT on an agitator and left to settle at RT for 2 h. Plates were counted on a 2450 MicroBeta2 microplate counter (PerkinElmer). Each independent experiment was performed in triplicates. The experiments were performed with dDAT from two independent purifications.
3h nisoxetine
Manufactured by PerkinElmer
Sourced in United States
[3H]nisoxetine is a tritium-labeled compound used as a radioligand in scientific research. It is a selective norepinephrine transporter (NET) inhibitor. [3H]nisoxetine can be used in binding assays and autoradiography studies to investigate the distribution and function of the NET in various tissues and experimental models.
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Lab products found in correlation
3h citalopram, by PerkinElmer (2 mentions)
Tramadol, by Merck Group (1 mentions)
Duloxetine, by Merck Group (1 mentions)
Prism v5, by GraphPad (1 mentions)
Cu ysi beads, by PerkinElmer (1 mentions)
Prism 7, by GraphPad (1 mentions)
Clear bottom 96 well plate, by Corning (1 mentions)
Microbeta2 2450 microplate counter, by PerkinElmer (1 mentions)
Nomifensine, by Merck Group (1 mentions)
3h 5 ht, by PerkinElmer (1 mentions)
Paroxetine, by Merck Group (1 mentions)
3h win35 428, by PerkinElmer (1 mentions)
Ultima gold scintillation cocktail, by PerkinElmer (1 mentions)
Desipramine, by Merck Group (1 mentions)
Fluoxetine, by Merck Group (1 mentions)
3h 8 oh dpat, by PerkinElmer (1 mentions)
N 2 chloroethyl n ethyl 2 bromobenzylamine hydrochloride dsp4, by Merck Group (1 mentions)
35 s datp, by PerkinElmer (1 mentions)
Gf b filter, by Cytiva (1 mentions)
Prism 4, by GraphPad (1 mentions)
9 protocols using 3h nisoxetine
1
Na⁺-Dependent [³H]Nisoxetine Binding to dDAT
2
Binding Affinity Determination of Monoamine Transporters
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Binding assays were performed with 20 nM of purified dDAT protein by scintillation proximity assay. For determining nisoxetine Kd, [3H] Nisoxetine (Perkin Elmer) (in 1:5 molar ratio) was used in the range of 0.1 nM to 500 nM with 100 µM desipramine added to the control samples. Competition assays were done with 50 nM [3H] Nisoxetine (in 1:5 molar ratio) with the concentration range for tramadol (Sigma Aldrich) being 100 nM to 3 mM, duloxetine (Sigma Aldrich) from 0.1 nM to 30 µM and milnacipran (Sigma Aldrich) from 1 nM to 100 µM. The assays were done in 1 mM DDM, 0.2 mM CHS, 20 mM Tris-Cl (pH 8), 300 mM NaCl and 5% glycerol. The background values were subtracted to plot the final curves in GraphPad prism v5.0.1 and the Kd values were calculated. The IC50 values obtained from binding competition assays were used to deduce the Ki values by Cheng–Prusoff’s equation.
3
Dopamine Transporter Binding Assay
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The activity of purified dDAT was assessed by scintillation proximity assay (SPA). 50 ng per well (7 nM) of protein was incubated with 1.25 mg ml−1 copper yttrium silicate (Cu-YSi) beads (PerkinElmer) in buffer A supplemented with 200 mM NaCl. Nisoxetine saturation binding was set up using 10% [3H]nisoxetine (79.8 Ci mmol−1; PerkinElmer). DA competition binding assays was performed with 30 nM [3H]nisoxetine and increasing concentration of unlabeled DA. Non-specific binding was determined in the presence of 100 µM unlabelled nortriptyline. SPA experiments were set up in triplicate wells of a 96-well white wall clear bottom plate. The samples were incubated for 30 min at room temperature. The samples were further incubated for 16 h incubation at 4 °C for nisoxetine saturation binding experiments. [3H]nisoxetine binding was monitored using a MicroBeta liquid scintillation plate counter (PerkinElmer) using a 1-min counting protocol. Data were analyzed by non-linear regression analysis and fitted to a one-site saturation or dose-response function, respectively, using GraphPad Prism 7 software (GraphPad, San Diego, CA).
4
Radioligand Binding Assay Protocol
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Albiflorin, obtained from Beijing Wonner Biotech Co., Ltd. (China), has a molecular formula of C23H28O11 and a molecular weight of 480.46. [3H]-citalopram, [3H]-WIN35,428, [3H]-nisoxetine, [3H]5-HT, [3H]-NE and [3H]-DA were purchased from PerkinElmer Life Sciences (NEN, Boston, MA). Scintillation cocktail (Ultima Gold, catalogue number 6013329) was purchased from PerkinElmer Life and Analytical Sciences. Dulbecco’s modified Eagle’s medium and foetal bovine serum were purchased from Invitrogen Inc. (Grand Island, NY) and HyClone Corp. (South Logan, UT), respectively. Desipramine (catalogue number D-3900), fluoxetine (catalogue number F-132), paroxetine (catalogue number P-1372) and nomifensine (catalogue number M-2017) were purchased from Sigma-Aldrich (St. Louis, MO). All other radioligands were purchased from PerkinElmer Life and Analytical Sciences.
5
Autoradiographic Binding Assays of 5-HT1A, SERT, NET
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The autoradiographic binding assays for 5-HT1AR, SERT and norepinephrine transporter were performed using the following radioligands: (a) [3H]-8-OH-DPAT (233 Ci mmol−1), (b) [3H]-citalopram (70 Ci mmol−1) and (c) [3H]-nisoxetine (85 Ci mmol−1), respectively (Perkin-Elmer, Madrid, Spain) as described previously.17 The experimental conditions are summarized in Supplementary Table S2 . For 5-HT1AR-stimulated [35S]GTPγS autoradiography, coronal dorsal raphe nucleus (DR) sections were labeled with 0.04 nM [35S]GTPγS.17 Details are shown in Supplementary Information .
6
Radioligand Binding Assay Protocol
7
Rat Brain Membrane Binding Assay
8
Radioligand Binding Assay for NET
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For NET binding assays, [3H]nisoxetine binding was performed
essentially as described by Tejani-Butt et al.60 (link) The cortical membrane pellet was resuspended in 50 mM Tris-HCl
buffer, pH 7.4, containing 300 mM NaCl and 5 mM KCl (T2 buffer). The
binding assay was performed incubating aliquots of membranes (0.2–0.3
mg of protein) in T2 buffer with 1 nM [3H]nisoxetine (specific
activity, 80 Ci mmol–1; PerkinElmer Life Science)
in a final volume of 0.5 mL. Incubation was carried out at 4 °C
for 4 h. Nonspecific binding was defined in the presence of 10 μM
desipramine. Specific binding was obtained by subtracting nonspecific
binding from total binding and approximated to 85–90% of total
binding. The binding reaction was quenched by filtration through Whatman
GF/C glass-fiber filters using a Brandel Harvester. Filters were washed
four times with 5 mL of the ice-cold binding buffer and placed in
vials with 4 mL of a scintillation cocktail. Radioactivity was measured
by means of a β-counter.
NET binding parameters (maximal
binding capacity, Bmax, fmol/mg protein;
dissociation constant, Kd, nM) were evaluated
in rabbit cortical membranes by measuring specific binding of [3H]nisoxetine
at increasing concentrations of the radioligand.
essentially as described by Tejani-Butt et al.60 (link) The cortical membrane pellet was resuspended in 50 mM Tris-HCl
buffer, pH 7.4, containing 300 mM NaCl and 5 mM KCl (T2 buffer). The
binding assay was performed incubating aliquots of membranes (0.2–0.3
mg of protein) in T2 buffer with 1 nM [3H]nisoxetine (specific
activity, 80 Ci mmol–1; PerkinElmer Life Science)
in a final volume of 0.5 mL. Incubation was carried out at 4 °C
for 4 h. Nonspecific binding was defined in the presence of 10 μM
desipramine. Specific binding was obtained by subtracting nonspecific
binding from total binding and approximated to 85–90% of total
binding. The binding reaction was quenched by filtration through Whatman
GF/C glass-fiber filters using a Brandel Harvester. Filters were washed
four times with 5 mL of the ice-cold binding buffer and placed in
vials with 4 mL of a scintillation cocktail. Radioactivity was measured
by means of a β-counter.
NET binding parameters (maximal
binding capacity, Bmax, fmol/mg protein;
dissociation constant, Kd, nM) were evaluated
in rabbit cortical membranes by measuring specific binding of [3H]nisoxetine
at increasing concentrations of the radioligand.
9
NET Binding Assay in Brain Regions
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Mice were decapitated and brain regions, including the raphe nucleus, ventral tegmental area, dorsal striatum, and locus coeruleus were dissected on ice and homogenized with 25 ml of ice cold buffer containing 50mM tris, 5mM MgCl2, pH 7.4. Homogenates were centrifuged for 20min at 15 000 x g. The pellet was resuspended and centrifuged under the same condition three times. To the final suspension (0.2-0.6 mg/ml) was added for one hour. [ 3 H]nisoxetine (21.3Ci/mmol; Perkin Elmer; USA) and (1nM-10µM) imipramine were used for NET binding. The process was terminated by immersing the tubes in ice cold buffer followed by rapid filtration through Whatman GF/B filters. Radioactivity was measured using liquid scintillation counting. Binding data were analyzed using the iterative non-linear fitting software GraphPad Prism 4.0 to estimate dissociation constants (K D ) and maximum number of sites (B max ).
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