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).
Cu ysi beads
Manufactured by PerkinElmer
Cu-YSi beads are a type of lab equipment used for various applications. They are composed of a copper (Cu) core and a yttrium-stabilized silica (YSi) shell. The core-shell structure of the Cu-YSi beads provides unique properties and functionalities that can be utilized in different laboratory settings. The detailed description and intended use of the Cu-YSi beads are not available in an unbiased and factual format.
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Lab products found in correlation
5 protocols using cu ysi beads
1
Dopamine Transporter Binding Assay
2
VMAT2 Ligand Binding Assay
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Purified VMAT2 (wild-type eGFP tagged and VMAT2 chimera) were diluted to 5 nM in 1 mM DDM, 0.125 mM CHS in 20 mM Tris pH 8.0, 150 mM NaCl with 1 mg/ml CuYSi beads (Perkin Elmer). Protein was then mixed 1:1 to a final protein concentration of 2.5 nM in detergent buffer containing serially diluted 3H-labeled DTBZ (American Radiolabeled Chemicals) starting at 60 nM final concentration. Counts were then measured using a Microbeta2 scintillation counter in 96 well plates with triplicate measurements70 (link). Specific counts were obtained by subtracting values obtained by the addition of 100 µM reserpine. Mutants were evaluated similarly from cell lysates of transfected cells. Data were fit to a single-site binding equation using Graphpad Prism.
Competition binding experiments were performed at the same protein concentration in the same detergent buffer. 10 nM 3H-labeled DTBZ was added to buffer and used for nine 1:1 serial dilutions with detergent buffer which initially contained 100 µM reserpine (10 µM for chimera). Measurements were done in triplicates and fit with a one-site competitive binding equation in Graphpad Prism.
Competition binding experiments were performed at the same protein concentration in the same detergent buffer. 10 nM 3H-labeled DTBZ was added to buffer and used for nine 1:1 serial dilutions with detergent buffer which initially contained 100 µM reserpine (10 µM for chimera). Measurements were done in triplicates and fit with a one-site competitive binding equation in Graphpad Prism.
3
NMDAR Binding Affinity Assay
4
Strychnine Binding Affinity via SPA
5
Radioligand Binding Assay for VMAT2
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Purified VMAT2 (wild-type eGFP tagged and VMAT2 chimera) were diluted to 5 nM in 1 mM DDM, 0.125 mM CHS in 20 mM Tris pH 8.0, 150 mM NaCl with 1 mg/ml CuYSi beads (PerkinElmer). Protein concentration was estimated using FSEC and a standard GFP concentration curve. Protein was then mixed 1:1 to a final protein concentration of 2.5 nM in detergent buffer containing serially diluted 3H-labeled DTBZ (American Radiolabeled Chemicals) starting at 60 nM final concentration. Counts were then measured using a Microbeta2 scintillation counter in 96-well plates with triplicate measurements (Green et al., 2015 (link)). Specific counts were obtained by subtracting values obtained by the addition of 100 µM reserpine. Mutants were evaluated similarly from cell lysates of transfected cells. Data were fit to a single-site binding equation using GraphPad Prism.
Competition binding experiments were performed at the same protein concentration in the same detergent buffer. 10 nM 3H-labeled DTBZ was added to buffer and used for nine 1:1 serial dilutions with detergent buffer which initially contained 100 µM reserpine (10 µM for chimera). Measurements were done in triplicates and fit with a one-site competitive binding equation in GraphPad Prism.
Competition binding experiments were performed at the same protein concentration in the same detergent buffer. 10 nM 3H-labeled DTBZ was added to buffer and used for nine 1:1 serial dilutions with detergent buffer which initially contained 100 µM reserpine (10 µM for chimera). Measurements were done in triplicates and fit with a one-site competitive binding equation in GraphPad Prism.
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