HTS023M
ChemiSCREEN CRF1 Membrane Preparation
Human CRF1 GPCR membrane preparation for Radioligand binding Assays & GTPgammaS binding.
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About This Item
UNSPSC Code:
41106514
eCl@ss:
32161000
NACRES:
NA.84
biological source
human
recombinant
expressed in Chem-1 cells
manufacturer/tradename
ChemiScreen
Chemicon®
technique(s)
ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable
NCBI accession no.
UniProt accession no.
shipped in
dry ice
Gene Information
human ... CRHR1(1394)
General description
Full-length Human CRF1
The CRF1 receptor is a Gs-coupled GPCR expressed in the brain and pituitary gland that binds to several neuropeptides, including corticotropin-releasing factor (CRF) and urocortin, and the amphibian peptide sauvagine (Chen et al., 1993; Dautzenberg and Hauger, 2002; Bale and Vale, 2004). CRF plays a predominant role in stress response mediated by the hypothalamic-pituitary-adrenal axis, and alterations in CRF and its receptors CRF1 and CRF2 appear to be linked to depression and anxiety (Holsboer, 1999; Bale and Vale, 2004). A number of small molecule antagonists of the CRF1 receptor have been characterized, including R121919, SC241, NBI27914, antalarmin, DMP-696, and CP 154,526. When delivered in animal models of psychiatric disorders, these antagonists display effectiveness in reducing stress-related behaviors (Kehne and De Lombaert, 2002). Chemicon′s CRF1 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 CRF1 interactions with its ligands. The membrane preparations exhibit a Kd of 1 nM for [125I]-sauvagine. With 2.5 μg/well CRF1 Membrane Prep and 0.5 nM [125I]-sauvagine, a greater than 12-fold signal-to-background ratio is obtained.
Application
Radioligand binding assay, and GTPgammaS binding.
Biochem/physiol Actions
GPCR Class: B
Protein Target: CRF1
Target Sub-Family: CRF Receptor
Quality
Signal:background and specific binding values obtained in a competition binding assay with CRF1 membrane preparation.
5 g/well
Signal:Background: 13.2
Specific Binding (cpm): 31785
5 g/well
Signal:Background: 13.2
Specific Binding (cpm): 31785
Specifications
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 (Costar 3605), 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°CRadioligand: [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 10-fold signal:background with 125I-labeled Sauvagine at 0.50 nM
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 (Costar 3605), 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°CRadioligand: [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 10-fold signal:background with 125I-labeled Sauvagine at 0.50 nM
Physical form
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.
Storage and Stability
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
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.
Storage Class
12 - Non Combustible Liquids
wgk_germany
WGK 2
flash_point_f
Not applicable
flash_point_c
Not applicable
Certificates of Analysis (COA)
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Find documentation for the products that you have recently purchased in the Document Library.
CRF and CRF receptors: role in stress responsivity and other behaviors
Bale, Tracy L and Vale, Wylie W
Annual Review of Pharmacology and Toxicology, 44, 525-557 (2004)
The CRF peptide family and their receptors: yet more partners discovered
Dautzenberg, Frank M and Hauger, Richard L
Trends in Pharmacological Sciences, 23, 71-77 (2002)
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