ChemiSCREEN EP₃ Prostanoid Receptor Membrane Preparation

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.

Radioligand binding assay and GTPS binding.

GPCR Class: A

Protein Target: EP3

Target Sub-Family: Prostanoid

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

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.

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.

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