Arginine vasopressin (AVP) is a 9 amino acid peptide that functions as an antidiuretic, vasoconstrictor and neurotransmitter. The three vasopressin receptors, V_1a, V_1b and V₂, are GPCRs; V_1a and V_1b couple to Gq and calcium release, whereas V₂ couples to Gs. V₂ expressed in renal collecting ducts plays an important role in regulating renal free water excretion (Birnbaumer, 2000). Mutations in V₂ result in X-linked nephrogenic diabetes insipidus, a syndrome in which the kidney is unable to concentrate urine, leading to dehydration and hypernatremia (Birnbaumer, 2001). Conversely, elevated levels of AVP lead to hyponatremia in the syndrome of inappropriate antidiuretic hormone secretion (SIADH), congestive heart failure or cirrhosis, and V₂ selective antagonists have been developed to treat these conditions (Verbalis, 2002). Chemicon′s V₂ 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 V₂ interactions and its ligands. The membrane preparations exhibit a Kd of 2.83 nM for [³H]-[Arg⁸] Vasopressin. With 3.5 nM [³H]-[Arg⁸] Vasopressin, 10 μg/well V₂ Membrane Prep yields greater than 8-fold signal-to-background ratio.

TRANSFECTION: Full-length human AVPR2 cDNA encoding V₂

Human V2 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay and GTPγS binding.

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]-[Arg8] Vasopressin (Perkin Elmer#:NET-800 )

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 8-fold signal:background with ³H labeled [Arg8] Vasopressin at 3.5 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.

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

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