HTS091M
ChemiSCREEN Human DP Prostanoid Receptor Membrane Preparation
Human DP 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 ... PTGDR(5729)
General description
Full-length human PTGDR cDNA encoding DP
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 PGD2 is produced by mast cells upon activation by allergens, and is present at high levels in allergic diseases. PGD2 binds to two receptors, DP and CRTH2. DP activates Gs to increase cAMP levels, and lack of DP results in reduced allergic response in animal models of bronchial asthma (Matsuoka et al., 2000). Chemicon′s DP 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 DP interactions and its ligands. The membrane preparations exhibit a Kd of 10.39 nM for [3H]-PGD2. With 8 nM [3H]-PGD2, 5 µg/well and 10 µg/well DP Membrane Prep typically yield greater than 4-fold signal-to-background ratio.
Application
Radioligand binding assay and GTPS binding.
Biochem/physiol Actions
GPCR Class: A
Protein Target: DP
Target Sub-Family: Prostanoid
Quality
Signal:background and specific binding values obtained in a competition binding assay with varying amounts of DP membrane prep:
Table 2. IC50 values for ligands obtained in a competition binding assay with DP membrane preparation.
SPECIFICATIONS: 1 unit = 10 µg
Bmax for [3H] PGD2 binding: 5.3 pmol/mg protein;
Kd for [3H] PGD2 binding: ~10.39nM
| 10 µg/well | 5 µg/well | |
|---|---|---|
| Signal:Background | 4.86 | 4.12 |
| Specific Binding (cpm) | 1345 | 885 |
Table 2. IC50 values for ligands obtained in a competition binding assay with DP membrane preparation.
| IC50 (nM) | |
|---|---|
| Prostaglanding D2 | 12.1 |
| 13,14-dihydro-15-keto prostaglandin D2 | 10000 |
| BW A868C | 4.8 |
SPECIFICATIONS: 1 unit = 10 µg
Bmax for [3H] PGD2 binding: 5.3 pmol/mg protein;
Kd for [3H] PGD2 binding: ~10.39nM
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 Hepes, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.2% BSA, filtered and stored at 4°C
Radioligand: [3H] PGD2 (Perkin Elmer#:NET-616 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, 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 4-fold signal:background with 3H-labeled PGD2 at 8 nM
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 Hepes, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.2% BSA, filtered and stored at 4°C
Radioligand: [3H] PGD2 (Perkin Elmer#:NET-616 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, 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 4-fold signal:background with 3H-labeled PGD2 at 8 nM
Physical form
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.
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
Certificates of Analysis (COA)
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T Matsuoka et al.
Science (New York, N.Y.), 287(5460), 2013-2017 (2000-03-17)
Allergic asthma is caused by the aberrant expansion in the lung of T helper cells that produce type 2 (TH2) cytokines and is characterized by infiltration of eosinophils and bronchial hyperreactivity. This disease is often triggered by mast cells activated
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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