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Assay Method Information

Assay Name:  D1R Functional Assay
Description:  The [35S]GTPγS binding assay was performed at 30 °C for 30 min containing 10 μgof membrane protein in a final volume of 100 μL with various concentration of the compound. The antagonism effects of the compounds were tested in the existence of10 μm haloperidol for the D1R. The binding buffer contains 50 mm Tris, pH 7.5, 5 mm MgCl2, 1 mm ethylenediamine tetraacetic acid (EDTA), 100 mm NaCl, 1 mm DL-dithiothreitol, and 40 μm guanosine triphosphate. The reaction was initiated by the addition of [35S]GTPγS (final concentration of 0.1 nm). Non-specificbinding was measured in the presence of 100 μm 50-guanylimidodiphosphate (Gpp(NH)p). The reaction was terminated by the addition of 1 mL of ice-coldwashing buffer (50 mm Tris, pH 7.5, 5 mm MgCl2, 1 mm EDTA, and 100 mm NaCl) and was rapidly filtered with GF/C glass fiber filters (Whatman) and washed three times. Radioactivity was determined by liquid scintillation counting. Again, l-SPDwas used as the control
Affinity data for this assay
 

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Last update November 1, 2007
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