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.
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