ChemiSCREEN Membrane Preparation Recombinant Human CRF ₂ Neuropeptide Receptor

Signal:background and specific binding values obtained in a competition binding assay with CRF₂ membrane preparation.
5 μg/well
Signal:Background 64.6
Specific Binding (cpm): 38718

SPECIFICATIONS: 1 unit = 5 μg
Bmax: 9.04 pmol/mg protein
Kd : 0.65 nM

Human CRF2 GPCR membrane preparation for Radioligand binding Assays & GTPgammaS binding.

Radioligand binding assay, and GTPgammaS binding.

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

RECOMMENDED ASSAY 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: 50 mM Hepes, pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 0.2% BSA, filtered and stored at 4°C

Radioligand: [¹²⁵I] Sauvagine (Perkin Elmer#:NEX306)

Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.

One vial contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 20-fold signal:background with ¹²⁵I-labeled Sauvagine at 0.35 nM

Full-length human CRHR2 cDNA encoding CRF₂

The corticotropin-releasing factor receptors, CRF₁ and CRF₂, are Gs-coupled GPCRs expressed in the brain, blood vessels and intestine that bind to several neuropeptides, including corticotropin-releasing factor (CRF) and urocortin, and the amphibian peptide sauvagine (Lovenberg et al., 1995; Bale and Vale, 2004). In addition, two peptides, urocortin II (Ucn II) and urocortin III (Ucn III), bind selectively and with high affinity to CRF₂ (Lewis et al., 2001). The CRF peptides and their receptors play important roles in stress mediated by the hypothalamic-pituitary-adrenal axis in animal models, and possibly in depression and anxiety in humans, although the contributions of CRF₁ and CRF₂ appear to be distinct (Bale and Vale, 2004; Risbrough et al., 2004) In addition, CRF₁ and CRF₂ differentially alter feeding behavior, gastric motility and vascular tone (Zorrilla et al., 2004; Martinez et al., 2004; Wiley and Davenport, 2004). Millipore′s CRF2 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 CRF₂ interactions with its ligands. The membrane preparations exhibit a Kd of 0.65 nM for [¹²⁵I]-sauvagine. With 5 μg/well CRF2 Membrane Prep and 0.35 nM [¹²⁵I]-sauvagine, a greater than 20-fold signal-to-background ratio is obtained.

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.

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