Description
General description
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
Application
Radioligand binding assay and GTPγS binding
Physical form
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.
Preparation Note
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Analysis Note
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.
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.
