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Figure 1. Saturation binding for CRF1. 10 µg/well CRF1 Membrane Preparation was incubated with increasing amount of ¹²⁵I-labeled S ...

Figure 2. Competition binding for CRF1. 5 µg/well CRF1 Membrane Preparation (HTS023M) was incubated with 0.50 nM ¹²⁵I-labeled Sau ...

ChemiSCREEN™ CRF1 Membrane Preparation

Description:

Trade Name:

ChemiScreen

Product Overview:

Full-length Human CRF1

Background Information:

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 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.

Host Cells:

Chem-1, an adherent mammalian cell line without any endogenous CRF1 expression.

Cell Type:

Chem-1

Gene Symbol:

CRHR1
CRF1
CRFR
CRHR1f
CRHR
CRFR1
CRF-R
CRH-R1h

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:

Signal:background and specific binding values obtained in a competition binding assay with CRF1 membrane preparation.

	5 µg/well
Signal:Background	13.2
Specific Binding (cpm)	31785

Protein or Enzyme Type:

Membrane Preparations

Incubation 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

Species:

Human

Storage Conditions:

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