ChemiSCREEN Membrane Preparation Recombinant Human CXCR1 Chemokine Receptor

: Full-length human CXCR1 cDNA

CXCR1 (also known as IL-8RA) is a Gi/o-coupled GPCR expressed on neutrophils, CD8(+) T cells, and intestinal epithelial cells (Murphy, 1997; Sturm et al., 2005; Takata et al., 2004). CXCR1 binds specifically to the chemokine interleukin-8 (IL-8), whereas a related receptor, CXCR2, binds promiscuously to IL-8 and several other chemokines. CXCR1 has been proposed to be the primary regulator of neutrophil response to IL-8 in sepsis (Cummings et al., 1999). Antagonists of CXCR1 and CXCR2 have been developed and display efficacy in animal models of reperfusion injury (Bertini, et al., 2004; Kaneider et al., 2005). Millipore′s CXCR1 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 CXCR1 interaction with IL-8. The membrane preparations exhibit a Kd of 0.2 nM for [¹²⁵I]-IL-8. With 5 μg/well CXCR1 Membrane Prep and 0.1 nM [¹²⁵I]-IL-8, a greater than 5-fold signal-to-background ratio is obtained.

Radioligand binding assay, and GTPgammaS binding.

GPCR Class: A

Protein Target: CXCR1 / IL-8a

Target Sub-Family: Chemokine

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, 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] Interleukin-8 (Perkin Elmer #NEX277)

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 5-fold signal:background with ¹²⁵I-labeled IL-8 at 0.1 nM

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.Packaging method: Membranes protein were adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.

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

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of CXCR1 Receptor membrane preparation in a dose response curve with unlabeled IL-8.

	5 µg/well	10 µg/well
Signal:Background	14.8	12.8
Specific Binding (cpm)	8965	9624

SPECIFICATIONS: 1 unit = 5 μg

Bmax: 3.7 pmol/mg;

Kd: 0.2 nM

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