ChemiScreen D₁ Dopamine Receptor Membrane Preparation

Dopamine exerts its action by binding to five distinct dopamine receptors, all of which belong to G protein-coupled receptor family (Missale et al. 1998). The D₁ subtype is the most abundant dopamine receptor in the central nervous system. Activation of D₁ receptor stimulates adenylyl cyclase, activates cyclic AMP-dependent protein kinases. It regulates neuronal growth and development, mediate some behavioral responses and modulate dopamine receptor D₂-mediated events. Chemicon′s D₁ membrane preparations are crude membrane preparations made from our proprietary stable recombinant cell lines to ensure high-level of GPCR surface expression; thus, they are ideal HTS tools for screening of antagonists of H₁ interactions and its ligands. The membrane preparations exhibit a Kd of 1.5 nM for [^{3H]-SCH 23390. With 1.5 nM [3H]- SCH 23390, 2.5 µ}g/well and 10 µg/well D₁ Membrane Prep typically yield greater than 5 and 15-fold signal-to-background ratios, respectively.

Full-length human DRD1 cDNA

Radioligand binding assay and GTPS binding.

GPCR Class: A

Protein Target: D1

Target Sub-Family: Dopamine

Bmax: 11.85 pmol/mg; Kd: 1.5 nM

Inucbation Conditions
Membranes are mixed with radioactive ligand and unlabeled competitor (see Figures 1 and 2 for concentrations tested) in binding buffer in a nonbinding 96-well plate, and incubated for 1-2 h. Prior to filtration, a GF/C 96-well filter plate is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM HEPES, pH 7.4, 0.5% BSA. Binding reaction is transferred to the filter plate, and washed 3 times (1 mL per well per wash) with Wash Buffer. The plate is dried and counted.
Binding buffer: 50 mM Tris, pH 7.4, 10 mM MgCl₂, 1 mM EDTA, filtered and stored at 4°C
Radioligand: [³H] SCH 23390 (Perkin Elmer#:NET-930 )
Wash Buffer: 50 mM Tris, pH 7.4 filtered and stored at 4°C.
One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 10-fold signal:background with ³H-labeled SCH 23390 at 1.5 nM

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 0.5 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C.

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

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