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Figure 1. Saturation binding for CXCR1. 10 µg/well CXCR1 Membrane Preparation was incubated with increasing amount of 125I-labeled ...
Figure 2. Competition binding for CXCR1. 10 µg/well CXCR1 Membrane Preparation (HTS001M) at 5 and 10 µg/well and Wild-Type-Chem-1 Membrane Pr ...
ChemiSCREEN™ Membrane Preparation Recombinant Human CXCR1 Chemokine Receptor
Description:
ChemiSCREEN™ Membrane Preparation Recombinant Human CXCR1 Chemokine Receptor
Trade Name:
ChemiScreen
Product Overview:
: Full-length human CXCR1 cDNA
Background Information:
CXCR1 (also known as IL-8RA) is a Gi/o-coupled GPCR expressed on neutrophils, CD8(+) T cells, and intestinal epithelial cells (Murphy, 1997; Sturm et al., 2005; Takata et al., 2004). CXCR1 binds specifically to the chemokine interleukin-8 (IL-8), whereas a related receptor, CXCR2, binds promiscuously to IL-8 and several other chemokines. CXCR1 has been proposed to be the primary regulator of neutrophil response to IL-8 in sepsis (Cummings et al., 1999). Antagonists of CXCR1 and CXCR2 have been developed and display efficacy in animal models of reperfusion injury (Bertini, et al., 2004; Kaneider et al., 2005). Millipore's CXCR1 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 HTS tools for screening of antagonists of CXCR1 interaction with IL-8. The membrane preparations exhibit a Kd of 0.2 nM for [125I]-IL-8. With 5 µg/well CXCR1 Membrane Prep and 0.1 nM [125I]-IL-8, a greater than 5-fold signal-to-background ratio 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.
Concentration:
1 mg/mL
Host Cells:
Chem-1, an adherent cell line expressing the promiscuous G-protein, Gα15 and lacking endogenous expression of CXCR1 or CXCR2.
Cell Type:
Chem-1
Gene Symbol:
- IL8RA
- CMKAR1
- CKR-1
- C-C
- C-C-CKR-1
- CXCR-1
- IL8RBA
- IL8R1
- CDw128a
- CD128
- CXCR1
- CD181
Format:
Membranes
Presentation:
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.Packaging method: Membranes protein were adjusted to the indicated concentration in 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 varying amounts of CXCR1 Receptor membrane preparation in a dose response curve with unlabeled IL-8.
SPECIFICATIONS: 1 unit = 5 µg
Bmax: 3.7 pmol/mg;
Kd: 0.2 nM
| 5 µg/well | 10 µg/well | |
|---|---|---|
| Signal:Background | 14.8 | 12.8 |
| Specific Binding (cpm) | 8965 | 9624 |
SPECIFICATIONS: 1 unit = 5 µg
Bmax: 3.7 pmol/mg;
Kd: 0.2 nM
Protein or Enzyme Type:
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, 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] Interleukin-8 (Perkin Elmer #NEX277)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM 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 5-fold signal:background with 125I-labeled IL-8 at 0.1 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] Interleukin-8 (Perkin Elmer #NEX277)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM 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 5-fold signal:background with 125I-labeled IL-8 at 0.1 nM
Species:
Human
Storage Conditions:
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.