ChemiSCREEN Membrane Preparation Recombinant Human NMU2 Neuromedin U Receptor

Human NMU2

Neuromedin U (NmU) is a peptide which regulates peripheral functions such as smooth muscle contraction and blood pressure, and CNS functions including nociception and feeding activity (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). Compared to the wide distribution of NMU1 in peripheral tissue, expression of NMU2 receptor is limited to areas of the brain, such as the paraventricular nucleus, along the wall of the third ventricle in the hypothalamus and the CA1 region of the hippocampus, and to the spinal cord (Howard et al. 2000). Recent study has shown that mice deficient in NMU2 but not NMU1 receptor had impaired nociceptive responses suggesting that the pro-nociceptive effects of NmU in mice appear to be mediated through NMU2 (Zeng et al., 2006; Torres et al. 2007). Millipore′s NMU2 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 0.58 nM for [¹²⁵I]-neuromedin U-25. With 5 μg/well NMU2 Membrane Prep and 0.25 nM [¹²⁵I]-neuromedin U-25, a greater than 10-fold signal-to-background ratio was obtained.

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: Membranes protein were adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.

Store at –70°C. Product is stable for at least 6 months from the date of receipt when stored as directed. Do not freeze and thaw.

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, 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]-neuromedin U-25 (Perkin Elmer # NEX383)

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 10-fold signal:background with 125I-labeled neuromedin U-25 at 0.25 nM.

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.