ChemiSCREEN CX3CR1 Membrane Preparation

CX3CR1 is a GPCR expressed on natural killer cells, cytotoxic T lymphocytes, and macrophages. The sole ligand for CX3CR1, fractalkine, is an unusual chemokine that is expressed as a transmembrane molecule with a CX3C domain and a mucin domain (Imai et al., 1997). Fractalkine is highly expressed on endothelial cells activated by TNFa and other proinflammatory cytokines, and fractalkine/CX3CR1 interactions mediate recruitment of macrophages into the atherosclerotic plaque (Lesnick et al., 2003; McDermott et al., 2003). In addition, fractalkine and CX3CR1 have been implicated in the pathogenesis of glomerulonephritis, HIV infection and rheumatoid arthritis (Ito et al., 2002; Faure et al., 2003; Nanki et al., 2002). Chemicon′s CX3CR1 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 fractalkine/CX3CR1 interactions. The membrane preparations exhibit a Kd of 0.05 nM for [¹²⁵I]-fractalkine. With 2.5 mg/well CX3CR1 Membrane Prep and 0.05 nM [¹²⁵I]-fractalkine, a greater than 7-fold signal-to-background ratio is obtained.

Full-length Human CX3CR1

Human CX3CR1 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay, and GTPγS binding.

GPCR Class: A

Protein Target: CX3CR1

Target Sub-Family: Chemokine

Bmax: 3.13 pmol/mg

Kd: 0.05 nM

Inucbation Conditions
RECOMMENDED ASSAY CONDITIONS:

Binding buffer: 50mM HEPES, 5mM MgCl₂, 1mM CaCl₂, 0.2% BSA, pH to 7.4 and filter.

Radioligand: [¹²⁵I] Fractalkine (PerkinElmer#: NEX368)

One vial contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 5-fold signal:background with ¹²⁵I-labeled fractalkine at 0.05 nM.

Packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA

Packaging method: Membranes protein were adjusted to 1 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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