CHEMISCREEN(TM) MEMBRANE PREP HU RECOMBINANT CXCR3

General description

CXCR3 is a 7-TM GPCR that is selective for the CXC chemokines IP10, ITAC and MIG (Loetscher et al., 1996). Binding of IP10 and MIG to CXCR3 induces Ca²⁺ mobilization, chemotaxis and inflammatory responses of T lymphocytes, and also act as potent inhibitors of angiogenesis. CXCR3 is highly expressed in IL-2-activated T lymphocytes in vitro (Loetscher et al., 1996), and in T lymphocytes present in inflamed tissues in rheumatoid arthritis and multiple sclerosis (Balashov et al., 1999; Qin et al., 1998). In vivo, neutralization of CXCR3 inhibits experimentally induced type I diabetes (Frigerio et al., 2002), peritonitis (Xie et al., 2003), and post-lung transplantation bronchiolitis obliterans syndrome (Belperio et al., 2002). Millipore’s ChemiScreenTM CXCR3 receptor membrane preparations are ideal tools for screening for antagonists of CXCR3 interactions with its ligands. With 0.1 nM ¹²⁵I-labeled IP10, the CXCR3 membrane preparation at 5 μg/well gives a signal:bakground of greater than 10-fold.

TRANSFECTION: Full-length human CXCR3 cDNA

Biochem/physiol Actions

Protein Target: CXCR3

Quality

Table 1. Signal:background and specific binding values obtained in a competition binding assay with CXCR3 membrane prep.

	10 µg/well
Signal:Background	28.2
Specific Binding (cpm)	6028

SPECIFICATIONS: 1 unit = 5 µg
Bmax for [¹²⁵I]IP-10 binding: 0.6 pmol/mg protein
Kd for [¹²⁵I]IP-10 binding: ~ 0.2 nM

Specifications

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] IP10 (Perkin Elmer #NEX348)

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 10-fold signal:background with ¹²⁵I-labeled IP10 at 0.1 nM

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CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

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