ChemiSCREEN Membrane Preparation Recombinant Human MCH₁ Receptor

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]-MCH (Perkin Elmer NEX373)

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

Radioligand binding assay and GTPγS binding

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Full length human MCHR1 cDNA encoding MCH₁

Melanin-concentrating hormone (MCH) is a cyclic, 19 amino acid peptide produced in the lateral hypothalamus and other regions of the mammalian brain (Bittencourt et al., 1992). When administered intracerebrally or overexpressed transgenically in rodents, MCH stimulates food intake and weight gain (Ludwig et al., 2001). A receptor for MCH, previously identified as an orphan receptor SLC-1 and renamed MCH₁, has been identified in humans and rodents. Consistent with a role in hypothalmic control of feeding behavior, MCH₁ is expressed in in the ventromedial and dorsomedial nuclei of the hypothalamus (Chambers et al., 1999). Genetic ablation and pharmacological antagonism of MCH₁ in rodents results in resistance to obesity caused by a high fat diet or leptin deficiency (Borowsky et al., 2002; Segal-Lieberman, et al., 2003; Shearman et al., 2003; Marsh et al., 2002; Chen et al., 2002). Thus, MCH₁ is an attractive target for obesity. Millipore′s MCH₁membrane preparations are crude membrane preparations made from our proprietary stable recombinant cell lines to ensure high-level GPCR surface expression; thus, they are ideal HTS tools for screening for agonists and antagonists of MCH₁. The membrane preparations exhibit a Kd of 0.41 nM for [¹²⁵I]-MCH. With 0.4 nM for [¹²⁵I]-MCH, 5 μg/well MCH₁ Membrane Prep yields greater than 8 fold signal-to-background ratio.

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 8-fold signal:background with ¹²⁵I-labeled MCH at 0.4 nM.

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.

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.