ChemiSCREEN CRTH2 Membrane Preparation

General description

Chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2) is a GPCR related to FPR-like chemoattractant receptors that is highly expressed on Th2 cells, eosinophils, basophils, and type 2 cytotoxic T cells (Nagata et al., 1999). The most potent ligand for CRTH2 is the prostaglandin PGD₂, a major mast cell-derived allergic mediator (Hirai et al., 2001). Another GPCR, DP, is activated by PGD₂, and the relative contributions of DP and CRTH2 to PGD₂-mediated allergic disease are being elucidated. A small molecule antagonist of CRTH2, ramatroban, attenuates PGD₂-mediated bronchial hyperresponsiveness and antigen-induced inflammation (Sugimoto et al., 2003; Shichijo et al., 2003).Chemicon′s CRTH2 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 PGD₂/CRTH2 interactions. The membrane preparations exhibit a Kd of 10 nM for [³H]-Prostaglandin D₂. With 2.5 μg/well CRTH2 Membrane Prep and 10 nM [³H]-Prostaglandin D₂, a greater than 5-fold signal-to-background ratio is obtained.

Human CRTH2

Application

Radioligand binding assay, and GTPgammaS binding.

Biochem/physiol Actions

GPCR Class: A

Protein Target: CRTH2 / DP2 / GPR44

Target Sub-Family: Prostanoid

Packaging

1 mL

Quality

Signal:background and specific binding values obtained in a competition binding assay with CRTH2 membrane preparation with unlabeled Prostaglandin D₂.
10 g/well
Signal:Background 12.2
Specific Binding (cpm) 4307

IC50 values for ligands obtained in a competition binding assay with CRTH2 membrane preparation.
IC50 (nM) Prostaglanding D₂ 4.5
13,14-dihydro-15-keto prostaglandin D₂ 14.7
BW A868C 7429

SPECIFICATIONS: 1 unit = 10 g
Bmax: 13.93 pmol/mg
Kd: 10 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, an FC 96-well harvest plate (Millipore cat. # MAHF C1H) 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: 50mM HEPES, 10mM MnCl2, 1mM EDTA, 0.2% BSA, pH to 7.0 and filter.
Wash buffer: 50mM HEPES, 0.5M NaCl, pH to 7.4 and filter.
Radioligand: [3H] Prostaglandin D2 (Amersham#: TRK734)

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.

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.