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Figure 1. Saturation binding for CXCR4. 5 µg/well CXCR4 Membrane Preparation was incubated with increasing amount of 125I-labeled ...
Figure 2. Competition binding for CXCR4. 5 µg/well CXCR4 Membrane Preparation was incubated with 125I-labeled SDF-1α (0.065, 1.3 o ...
ChemiSCREEN™ CXCR4 Membrane Preparation
Description:
ChemiSCREEN™ CXCR4 Membrane Preparation
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Product Overview:
Full-length human CXCR4 cDNA
Background Information:
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 Notes:
Radioligand binding assay and GTPgammaS binding.
Species Reactivity:
Human
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Cell Membrane Type:
GPCR
Descriptive Text:
Concentration:
2 mL, 0.5 mg/mL
Host Cells:
Chem-1
Cell Type:
Chem-1
Gene Symbol:
- CXCR4
- WHIM
- NPY3R
- NPYY3R
- FB22
- LAP3
- Fusin
- CXCR-4
- LCR1
- NPYR
- HM89
- fusin
- HSY3RR
- CXC-R4
- NPYY3
- NPYRL
- D2S201E
- CD184
- LESTR
Format:
Membranes
Presentation:
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.
Packaging method: Membranes protein were adjusted to 0.5 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C.
Quality Assurance:
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
Protein or Enzyme Type:
GPCR Membrane Preparations
Incubation 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.
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.
Species:
Human
Storage Conditions:
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.