ChemiSCREEN Human δ Opioid Receptor Membrane Preparation

32161000

NA.84

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.
Full-length human OPRD1 cDNA encoding δ opioid receptor

Radioligand binding assay and GTPγS

Protein Target: Delta / OP1 / DOP / DOR
GPCR Class: A
Target Sub-Family: Opioid

GPCR Class: A

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.

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.

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

100

human

expressed in Chem-1 cells

ChemiScreen
Chemicon^®

ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable

NM_000911.3

P41143

dry ice

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.

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12 - Non Combustible Liquids

WGK 2