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General description
Full-length human OPRD1 cDNA encoding δ opioid receptor
Opiates derived from the opium poppy, Papaver somniferum, have been used in for millenia for their anti-diarrheal, analgesic and euphoric properties. More recently, endogenous peptides, enkephalins, dynorphins, and endorphins, were found to bind to the same sites as opiate alkaloids. The receptors for the classical opioids are three related GPCRs, δ, κ, and μ (also known as OP1, OP2 and OP3, resepctively), that activate Gi/o to reduce intracellular cAMP levels. Although most clinically used opioids function by activation of the µ opioid receptor, agonits of spinal δ opioid receptors have antinociceptive activity that is independent of µ. In addition, activation of δ increases locomotor activity, inhibits gastrointestinal motility, and decreases respiratory frequency (Dhawan et al., 1996). Agonists for δ opioid receptors also exhibit antidepressant-like activity in animal models (Broom et al., 2002). Chemicon′s δ 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 agonists and antagonists at δ. The membrane preparations exhibit a Kd of 1.12 nM for [3H]-DADLE. With 0.9 nM [3H]-DADLE, 10 µg/well δ Membrane Prep typically yields greater than 4-fold signal-to-background ratio.
Application
Radioligand binding assay and GTPγS
Biochem/physiol Actions
GPCR Class: A
Protein Target: Delta / OP1 / DOP / DOR
Target Sub-Family: Opioid
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-HCl. 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-HCl, filtered and stored at 4°C
Radioligand: [3H] DADLE (Perkin Elmer#: NET-648)
Wash Buffer: 50 mM Tris-HCl, filtered and stored at 4°C.
Physical form
One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 4-fold signal:background with 3H-labeled DADLE at 0.9 nM
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membranes protein were adjusted to 1 mg/mL in packaging buffer, and dispensed at 1 mL/vial. Vials were rapidly frozen, and stored at -80°C.
Preparation Note
Store at –70°C. Product is stable for at least 6 months from the date of receipt when stored as directed. Do not freeze and thaw.
Analysis Note
Signal:background and specific binding values obtained in a competition binding assay with δ membrane prep and unlabeled DPDPE.
10 g/well
Signal:Background: 7.32
Specific Binding (cpm): 955
SPECIFICATIONS: 1 unit = 10 g
Bmax for [3H] DADLE binding: 11.6 pmol/mg protein
Kd for [3H] DADLE binding: ~1.12 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_000911.3 |
| UniProt accession no. | P41143 |
| shipped in | dry ice |
| storage temp. | -65 to -96°C |
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