ChemiScreen Recombinant Human NMU1 Neuromedin Receptor

Human NMU1 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay, and GTPγS binding.

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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]-neuromedin U25 (Perkin Elmer # NEX383)

Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.

Human NMU1

Neuromedin U (NmU) is a peptide characterized by its ability to promote smooth muscle contraction (Brighton et al., 2004a). Two GPCRs, NMU1 and NMU2, mediate the contractile effects of neuromedin U by activation of both Gq and Gi (Brighton et al., 2004b). In addition, NmU binds to NMU1 expressed on Th2 cells to induce cytokine release (Johnson et al., 2004). Chemicon′s NMU1 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 NMU1 interactions with neuromedin U25. The membrane preparations exhibit a Kd of 0.08 nM for [¹²⁵I]-neuromedin U25. With 5 μg/well NMU1 Membrane Prep and 0.5 nM [¹²⁵I]-neuromedin U25, a greater than 30-fold signal-to-background ratio was obtained.

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 30-fold signal:background with ¹²⁵I-labeled neuromedin U25 at 0.5 nM.
Liquid in packaging buffer:

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

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