Description
General description
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 Gq 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.
Application
Human EP1 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.
Radioligand binding assay and GTPγS binding
Biochem/physiol Actions
GPCR Class: A
Protein Target: EP1
Target Sub-Family: Prostanoid
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 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.
Physical form
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.
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
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
Kd: 14.5 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.
