HTS018M
ChemiSCREEN FPR1 Membrane Preparation
Human FPR1 GPCR membrane preparation for Radioligand binding Assays & GTPgammaS binding.
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About This Item
UNSPSC Code:
41123201
eCl@ss:
32161000
NACRES:
NA.84
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General description
FPR1 is a Gi-coupled GPCR that binds and is activated by short, N-formyl peptides, such as N-Formyl-Met-Leu-Phe (fMLF). N-formyl peptides are typically derived from bacteria and mediate host recruitment of leukocytes, which highly express FPR1, to sites of bacterial infection (Gao et al., 1999). In addition, FPR1 is expressed in the vasculature, secretory epithelial cells, neurons and other tissues (Becker et al., 1998). Other ligands, such as mitochondrially encoded formyl peptides, amyloid proteins, and virus-derived peptides, have also been found to activate FPR1 and/or related GPCRs and may be important in the pathophysiology of amyloidoses, Alzheimer′s disease and HIV (Le et al., 2002). A synthetic hexapeptide ligand, WKYMVm, has also been demonstrated to bind to FPR1 (Le et al., 1999). Chemicon′s 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 FPR1 and its ligands. The membrane preparations exhibit a Kd of 0.39 nM for [125I]-WKYMVm. With 5 μg/well FPR1 Membrane Prep and 0.5 nM [125I]-WKYMVm, a greater than 20-fold signal-to-background is obtained; with 1.25 and 2.5 μg/well FPR1 Membrane Prep, greater than 9- and 15-fold signal-to-background ratios are obtained, respectively.
Human FPR1
application
Radioligand binding assay, and GTPgammaS binding.
Biochem/physiol Actions
GPCR Class: A
Protein Target: FPR1
Target Sub-Family: N-formylpeptide
Quality
SPECIFICATIONS:
Signal:background and specific binding for FPR1 membrane preparation.
Bmax: 5.65 pmol/mg
Kd: 0.39 nM
Signal:background and specific binding for FPR1 membrane preparation.
| 5 µg/well | |
|---|---|
| Signal:Background | 17.9 |
| Specific Binding (cpm) | 17837 |
Bmax: 5.65 pmol/mg
Kd: 0.39 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, a GF/C 96-well filter plate 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] WKYMVm (Perkin Elmer# NEX386)
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 10-fold signal:background with 125I-labeled WKYMVm at 0.5 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, a GF/C 96-well filter plate 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] WKYMVm (Perkin Elmer# NEX386)
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 10-fold signal:background with 125I-labeled WKYMVm at 0.5 nM
Physical form
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.Packaging method: Membranes protein were adjusted to 0.5 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C.
Storage and Stability
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
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.
Storage Class
12 - Non Combustible Liquids
wgk_germany
WGK 2
flash_point_f
Not applicable
flash_point_c
Not applicable
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