HTS092M
ChemiSCREEN EP3 Prostanoid Receptor Membrane Preparation
Human EP3 GPCR membrane preparation for Radioligand binding Assays & GTPgammaS binding.
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About This Item
UNSPSC Code:
41106514
eCl@ss:
32161000
NACRES:
NA.41
biological source
human
Quality Level
recombinant
expressed in Chem-1 cells
manufacturer/tradename
ChemiScreen
Chemicon®
technique(s)
ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable
NCBI accession no.
UniProt accession no.
shipped in
dry ice
Gene Information
human ... PTGER3(5733)
General description
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.
Application
Radioligand binding assay and GTPS binding.
Biochem/physiol Actions
GPCR Class: A
Protein Target: EP3
Target Sub-Family: Prostanoid
Quality
Signal:background and specific binding values obtained in a competition binding assay with varying amounts of EP3 membrane prep:
Specifications:
1 unit = 10 μg
Bmax: 15.0 pmol/mg
Kd: 1.56 nM
| 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
Specifications
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.
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.
Physical form
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.
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.
Storage and Stability
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
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.
Storage Class Code
12 - Non Combustible Liquids
WGK
WGK 2
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
Regulatory Listings
Regulatory Listings are mainly provided for chemical products. Only limited information can be provided here for non-chemical products. No entry means none of the components are listed. It is the user’s obligation to ensure the safe and legal use of the product.
JAN Code
HTS092M:
Certificates of Analysis (COA)
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F Ushikubi et al.
Nature, 395(6699), 281-284 (1998-09-29)
Fever, a hallmark of disease, is elicited by exogenous pyrogens, that is, cellular components, such as lipopolysaccharide (LPS), of infectious organisms, as well as by non-infectious inflammatory insults. Both stimulate the production of cytokines, such as interleukin (IL)-1beta, that act
M Kotani et al.
Molecular pharmacology, 48(5), 869-879 (1995-11-01)
Five distinct cDNA clones encoding four different isoforms of human prostaglandin (PG) E receptor EP3 subtype were isolated from a human kidney cDNA library. Two cDNA clones differed only in their 3'-untranslated regions. The four isoforms, tentatively named EP3-I, EP3-II
M Kotani et al.
Genomics, 40(3), 425-434 (1997-03-15)
Prostaglandin EP3 receptor subtype is a seven-membrane-spanning protein with multiple C-terminal tails generated by alternative mRNA splicing. We report here the structural organization of the human EP3 gene (PTGER3). The human EP3 gene spanned more than 80 kb and was
Genetic and pharmacological analysis of prostanoid receptor function.
S Narumiya et al.
The Journal of clinical investigation, 108(1), 25-30 (2001-07-04)
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