ChemiSCREEN Human DP Prostanoid Receptor Membrane Preparation

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 G_s 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.

Radioligand binding assay and GTPS binding.

GPCR Class: A

Protein Target: DP

Target Sub-Family: Prostanoid

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

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.

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of DP membrane prep:

	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

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

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.