ChemiSCREEN Membrane Preparation Recombinant Human GPR103 / QRFP Neuropeptide Receptor

GPR103 is a Gq-coupled receptor that is located in the brain and adrenal gland, and is activated by a 43-amino acid RFamide peptide, QRFP43, and a 26 amino acid fragment, QRFP26/26RFa/Peptide 518 (Jiang et al., 2003; Fukusumi et al., 2003). QRFP43 is the active form in rat brain, and intracerebral administration of QRFP43 induces feeding and metabolic rate (Takayasu et al., 2006; Moriya et al., 2006). Millipore′s GPR103 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 GPR103. The membrane preparations exhibit a Kd of 0.63nM for [¹²⁵I]-QRFP43. With 10 μg/well GPR103 Membrane Prep and 0.35 nM [¹²⁵I]- QRFP43, a greater than 6-fold signal-to-background ratio was obtained.

Human GPR103

Radioligand binding assay and GTPγS binding

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membrane protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.

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

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]QRFP43 (Perkin Elmer # NEX408)

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 6-fold signal:background with ¹²⁵I-labeled QRFP43 at 0.35 nM.

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