ChemiSCREEN Membrane Preparation Recombinant Human EP₁ Prostanoid Receptor

Table 1. Signal:background and specific binding values obtained in a competition binding assay with EP₁ membrane prep.

	10 µg/well
Signal:Background	4.3
Specific Binding (cpm)	724

SPECIFICATIONS: 1 unit = 10 μg membrane preparation
Bmax: 4.6 pmol/mg
K_d: 14.5 nM

Human EP1 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay and GTPγS binding

GPCR Class: A

Protein Target: EP1

Target Sub-Family: Prostanoid

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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 10mM MES, pH 6, 1mM EDTA, 10mM MnCl₂. 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: 10mM MES, pH 6, 1mM EDTA, 10mM MnCl₂, filtered and stored at 4°C

Radioligand: [³H]- PGE₂. (Perkin Elmer # NET-428 )

Wash Buffer: 10mM MES, pH 6, 1mM EDTA, 10mM MnCl₂, 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 3-fold signal: background with ³H labeled PGE₂.

Full-length human PTGER1 cDNA encoding EP₁

Prostanoids are a series of arachidonic acid metabolites produced by the action of cyclooxygenase and subsequently by isomerases and synthases. Cells rapidly secrete prostanoids after synthesis, whereupon the 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; EP₁ couples primarily to G_q to mobilize intracellular calcium. EP₁ appears to mediate the effects of PGE₂ in promoting formation of precancerous lesions in animal models of colon cancer (Watanabe et al., 1999). In addition, EP₁ has an inhibitory effect on stress-induced aggressive and risk-taking behaviors in mice (Matsuoka et al., 2005). Millipore′s EP₁ 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 of EP₁. The membrane preparations exhibit a Kd of 14.5 nM for [³H]-PGE₂. With 40 nM [³H]-PGE₂, 10 μg/well EP₁ Membrane Prep typically yields greater than 3-fold signal-to-background ratio.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membrane protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.

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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