Description
General description
Dopamine is a catecholamine neurotransmitter that functions in the CNS to control locomotor, cognitive, emotional and neurendocrine processes, and in the periphery to modulate cardiovascular, renal and gastrointestinal processes. The biological activities of dopamine are mediated by a family of five GPCRs. The D₁ and D₅ subtypes couple to Gs to increase intracellular cAMP, whereas the D₂, D₃ and D₄ subtypes couple to Gi to reduce cAMP (Missale et al., 1998). The D₂ dopamine receptors have been of particular clinical interest due to their regulation of prolactin secretion and their affinity for antipsychotic drugs. The D₂ receptor exists as two alternatively spliced isoforms differing in the insertion of a stretch of 29 amino acids in the third intracellular loop (D₂S and D₂L) (Giros et al., 1989; Grandy et al., 1989). Millipore′s D₂ dopamine 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 D₂ dopamine receptor interactions with its ligand. The membrane preparations exhibit a Kd of 0.51nM for [¹²⁵I]-Iodospiperone. With 5.0 ug/well D2 Dopamine Membrane Prep and 0.1nM [¹²⁵I]-Iodospiperone, a greater than 10-fold signal-to-background ratio was obtained.
Human Dopamine Receptor 2
Application
Radioligand binding assay and GTPγS binding
Biochem/physiol Actions
GPCR Class: A
Protein Target: D2L
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, an FC 96-well harvest plate (Millipore cat. # MAHF C1H) is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM Tris, pH 7.4. 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, 120mM NaCl, filtered and stored at 4°C
Radioligand: [¹²⁵I] Iodospiperone (Perkin Elmer # NEX284)
Wash Buffer: 50 mM Tris, pH 7.4, filtered and stored at 4°C.
Physical form
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 [¹²⁵I]-labeled Iodospiperone at 0.1 nM.
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membranes protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.
Preparation Note
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Analysis Note
Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of Dopamine Receptor membrane prep. 10 µg/well 5 µg/well
Signal:Background 23.6 27.5
Specific Binding (cpm) 8331 9927.2
SPECIFICATIONS: 1 unit = 5.0 µg membrane preparation
Bmax: 6.03 pmol/mg
Kd: 0.51 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.
