HTS003M
ChemiSCREEN Membrane Preparation Recombinant Human CXCR3 Chemokine Receptor
Human CXCR3 GPCR membrane preparation for Radioligand binding Assays.
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About This Item
UNSPSC Code:
41106514
eCl@ss:
32161000
NACRES:
NA.41
Recommended Products
biological source
human
Quality Level
recombinant
expressed in Chem-1 cells
manufacturer/tradename
ChemiScreen
Chemicon®
concentration
1.0 mg/mL
technique(s)
radioligand binding assay (RLBA): suitable
NCBI accession no.
UniProt accession no.
shipped in
dry ice
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.
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
| 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
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
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Storage Class
12 - Non Combustible Liquids
wgk_germany
WGK 2
flash_point_f
Not applicable
flash_point_c
Not applicable
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