ChemiSCREEN EP₃ Prostanoid Receptor Membrane Preparation

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of EP₃ membrane prep:

	10 µg/well	5 µg/well
Signal:Background	38	21
Specific Binding (cpm)	6,386	4,053

Specifications:
1 unit = 10 μg
Bmax: 15.0 pmol/mg
Kd: 1.56 nM

Radioligand binding assay and GTPS binding.

GPCR Class: A

Protein Target: EP3

Target Sub-Family: Prostanoid

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.

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] PGE₂ (Perkin Elmer#:NET-428 )
Wash Buffer: 50 mM Tris, pH 7.4 filtered and stored at 4°C.

Full-length human PTGER₃ cDNA encoding splice variant 6 of EP₃

Prostanoids bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001). The prostaglandin PGE₂ causes pain, vasodilation, immunosuppression of T cells, bone resorption and promotion of carcinogenesis. Four related GPCRs, EP₁, EP₂, EP₃ and EP₄, each bind to PGE₂, but the different G protein coupling status of each receptor leads to distinct biological effects. Further diversity is generated by alternative splicing; the human gene for EP₃ generates 9 alternatively spliced mRNAs encoding 8 isoforms of EP₃ (Kotani et al., 1997). These isoforms of EP₃ vary in sequence at their C-termini, and differ in their ability to couple to Gs, Gq or Gi (Kotani et al., 1995). EP3 is required for fever induced by pyrogens, a response long attributed to prostaglandins by the antipyretic action of aspirin and other COX inhibitors (Ushikubi et al., 1998). Chemicon′s EP₃ 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 EP₃ interactions and its ligands. The membrane preparations exhibit a Kd of 1.56 nM for [3H]-PGE₂. With 1.5 nM [3H]-PGE₂, 5 µg/well and 10 µg/well EP₃ Membrane Prep typically yield greater than 10 and 20-fold signal-to-background ratios, respectively.

One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 10-fold signal:background with 3H-labeled PGE₂ 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.

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