Ordering Information
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Figure 1. Saturation binding for CRF2. 10µg/well CRF2 Membrane Preparation was incubated with increasing amount of 125I-labeled Sauvagine ...
Figure 2. Competition binding for CRF2. 5.0 µg/well CRF2 Membrane Preparation (HTS024M) was incubated with 0.35 nM 125I-labeled Sauvagin ...
ChemiSCREEN™ Membrane Preparation Recombinant Human CRF2 Neuropeptide Receptor
Description:
ChemiSCREEN™ Membrane Preparation Recombinant Human CRF2 Neuropeptide Receptor
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Product Overview:
Full-length human CRHR2 cDNA encoding CRF2
Background Information:
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.
Application Notes:
Radioligand binding assay, and GTPgammaS binding.
Species Reactivity:
Human
Usage Statement:
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.
Cell Membrane Type:
GPCR
Descriptive Text:
Concentration:
0.5 mg/mL
Host Cells:
Chem-1, an adherent mammalian cell line without any endogenous CRF2 expression.
Cell Type:
Chem-1
Gene Symbol:
- CRHR2
- CRH2R
- CRF2
- CRFR2
- CRF2R
Format:
Membranes
Presentation:
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.
Quality Assurance:
Table 1. Signal:background and specific binding values obtained in a competition binding assay with CRF2 membrane preparationn.
SPECIFICATIONS: 1 unit = 5 µg
Bmax: 9.04 pmol/mg protein
Kd : 0.65 nM
| 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
Protein or Enzyme Type:
GPCR Membrane Preparations
Incubation Conditions:
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
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
Species:
Human
Storage Conditions:
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.