ChemiSCREEN FPR1 Membrane Preparation

FPR1 is a G_i-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

Radioligand binding assay, and GTPgammaS binding.

GPCR Class: A

Protein Target: FPR1

Target Sub-Family: N-formylpeptide

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

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.

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

SPECIFICATIONS:

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

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