Description
General description
Full-length human CXCR4 cDNA
The chemokine SDF-1a and its GPCR receptor CXCR4 have a one-to-one specificity that is unique among chemokines and their receptors. SDF-1a binds to CXCR4 expressed on hematopoietic and lymphopoietic cells, and directs their trafficking to and retention in hemato-and lymphatopoietic organs and sites of inflammation (Kucia et al., 2004). CXCR4 is expressed on several tumor cell lineages, and might be responsible for metastasis to sites of SDF-1a expression, such as bone and lymph nodes (Muller et al., 2001). In addition, CXCR4 is a coreceptor for the HIV envelope glycoprotein gp120 (Feng et al., 1996). Small molecule antagonists of CXCR4 have been developed and shown to inhibit infectivity of T-tropic HIV strains and to impair growth of brain tumors (Arakaki et al., 1999; Rubin et al., 2003). Chemicon′s CXCR4 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 tools for screening for antagonists of interactions between CXCR4 and SDF-1a. Radiolabeled SDF-1a binds to the CXCR4 membranes with a Kd of 0.9 nM. With 0.04-0.13 nM 125I-labeled SDF-1a, an approximately 3-fold window of specific binding is obtained.
Application
Radioligand binding assay and GTPgammaS binding.
Biochem/physiol Actions
Protein Target: CXCR4
Features and Benefits
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 50 mM 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] SDF-1alpha (PerkinElmer#: NEX346)
Wash Buffer: 50 mM Hepes, pH 7.4, 500 mM 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 4-fold signal:background with 125I-labeled SDF-1alpha at 0.13 nM.
Physical form
Packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA
Packaging method: Membranes protein were adjusted to 0.5 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C.
Analysis Note
Table 1. Signal:background and specific binding values obtained in a competition binding assay with CXCR4 membrane prep, 0.13 nM 125I-SDF-1α and a dilution series of unlabeled SDF-1α.
5 µg/well
Signal:Background 4.9
Specific Binding (cpm) 9004
Specifications:
1 unit = 5 μg
Bmax [125I]-SDF-1alpha binding: 1.6 pmol/mg protein
Kd for [125I] SDF-1alpha binding: ~ 0.9 nM
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
