HTS004M

ChemiSCREEN CXCR4 Membrane Preparation

Human CXCR4 GPCR membrane preparation for Radioligand binding Assays & GTPgammaS binding.

• Code UNSPSC : 41106514
• eCl@ss : 32161000
• Nomenclature NACRES : NA.84

Propriétés

• Source biologique: human
• Niveau de qualité: 100
• Produit recombinant: expressed in Chem-1 cells
• Conditionnement: pkg of 2 mL
• Fabricant/nom de marque: ChemiScreen; Chemicon^®
• Concentration: 0.5 mg/mL
• Technique(s): ligand binding assay: suitable (GTPγS); radioligand binding assay (RLBA): suitable
• Numéro d'accès NCBI: NM_001008540.1; NM_003467.2
• Numéro d'accès UniProt: P61073
• Conditions d'expédition: dry ice
• Informations sur le gène: human ... CXCR4(7852)

Description

Description générale

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.

Actions biochimiques/physiologiques

Protein Target: CXCR4

Qualité

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

Caractéristiques

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.

Forme physique

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.

Informations légales

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

Informations sur la sécurité

• Code de la classe de stockage: 12 - Non Combustible Liquids
• Classe de danger pour l'eau (WGK): WGK 2
• Point d'éclair (°F): Not applicable
• Point d'éclair (°C): Not applicable