ChemiScreen CXCR6 Membrane Preparation

Human CXCR6 cDNA

The GPCR CXCR6 (previously known as BONZO, STRL33 and TYMSTR) binds selectively to the free chemokine domain of CXCL16, which is derived from a membrane-bound precursor containing a CXC-containing chemokine domain, a glycosylated mucin-like domain, and a transmembrane domain (Wilbanks et al., 2001) CXCR6 is selectively expressed on Th1, Th2 and Tr1 T cell subsets, whereas CXCL16 is expressed on monocytes/macrophages and dendritic cells (Tabata et al., 2005). CXCR6 promotes migration of activated lymphocytes to sites of inflammation in tissues such as liver and synovium (Nanki et al., 2005, Sato et al., 2005). Chemicon′s CXCR6 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 CXCR6 interactions with its ligands. The membrane preparations exhibit a Kd of 0.6 nM for [¹²⁵I]-CXCL16. With 2.5 or 10 μg/well CXCR6 Membrane Prep and 0.5 nM [¹²⁵I]-CXCL16, greater than 4-fold and 9-fold signal-to-background ratios are obtained, respectively.

Human CXCR6 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay, and GTPγS binding.

GPCR Class: A

Protein Target: CXCR6

Target Sub-Family: Chemokine

SPECIFICATIONS:
1 unit = 5 μg for GTPγS binding assay
EC50 in GTPγS binding assay by CXCL16: 0.52 – 1.2 nM

Inucbation 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, 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°C

Radioligand: ^[125I] CXCL16 (Perkin Elmer catalog # NEX398)

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

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 4-fold signal:background with ¹²⁵I-labeled CXCL16 at 0.5 nM

One vial contains enough membranes for at least 200 assays (units), where one unit is the amount of membrane that will yield greater than 1000 cpm specific CXCL16-stimulated [³⁵S]-GTPγS binding. If radioligand binding assays are desired, it is recommended to double the mass of membrane prep per well to increase signal window..

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.

Packaging method: Membrane protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

Store at –70°C. Product is stable for at least 6 months from the date of receipt when stored as directed. Do not freeze and thaw.

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