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General description
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 µg/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
Application
Radioligand binding assay and GTPS binding.
Biochem/physiol Actions
GPCR Class: A
Protein Target: D1
Target Sub-Family: Dopamine
Features and Benefits
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
Physical form
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.
Preparation Note
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Analysis Note
Bmax: 11.85 pmol/mg; Kd: 1.5 nM
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
| biological source | human |
| Quality Segment | 100 |
| recombinant | expressed in Chem-1 cells |
| manufacturer/tradename | ChemiScreen,Chemicon® |
| technique(s) | ligand binding assay: suitable (GTPγS),radioligand binding assay (RLBA): suitable |
| NCBI accession no. | NM_000794.3 |
| UniProt accession no. | P21728 |
| shipped in | dry ice |
| storage temp. | -65 to -96°C |
| Gene Information | human ... DRD1(1812) |
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