ChemiSCREEN GPR14 Membrane Preparation

TRANSFECTION: Full-length human GPR14 cDNA

Urotensin II, a cyclic 11-13 residue peptide expressed in motoneurons of the spinal cord, acts as a potent vasoconstrictor (Coulouarn et al., 1998; Ames et al., 1999). The effects of urotensin II are mediated by binding to a GPCR, GPR14, which is expressed in endothelium, smooth muscle, heart and pancreas (Ames et al., 1999). Genetic deletion of GPR14 in mice renders aortae refractile to the contractile activity of urotensin II without changing baseline hemodynamics (Behm et al., 2003). Pharmacological inhibition of urotensin II/GPR14 interactions prevents renal insufficiency following renal artery ligation (Clozel et al., 2004). Thus, GPR14 is an attractive target for a number of cardiovascular diseases. Chemicon′s GPR14 Membrane Preparations are ideal tools for screening for antagonists of urotensin II/GPR14 interactions. The membrane preparations exhibit a Kd of 0.4 nM for [125I]-urotensin II. With 10 mg/well GPR14 Membrane Prep and 0.3 nM [125I]-urotensin II, a greater than 20-fold signal-to-background ratio is obtained.

Human UT / UT1 / GPR14 GPCR membrane preparation for Radioligand binding Assays.

Radioligand binding assay.

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 50 mM 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.

Membranes were adjusted to 0.5 mg protein/ml in 50 mM Tris pH 7.4, 10% glycerol and 1% BSA, rapidly frozen, and stored at -80°C. One vial typically contains 2000 units, where a unit is the amount of membrane that will yield greater than 1000 cpm specific

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

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

RESEARCH USE ONLY. This product is regulated in France when intended to be used for scientific purposes, including for import and export activities (Article L 1211-1 paragraph 2 of the Public Health Code). The purchaser (i.e. enduser) is required to obtain an import authorization from the France Ministry of Research referred in the Article L1245-5-1 II. of Public Health Code. By ordering this product, you are confirming that you have obtained the proper import authorization.

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