ChemiSCREEN™ Membrane Preparat

ChemiSCREEN™ Membrane Preparation Recombinant Human MT₂ Melatonin Receptor

• ChemiScreen
• Chemicon (Millipore)

Full-length human MTNR1B cDNA encoding MT₂

Melatonin (5-methoxy-acetyltryptamine) is a hormone synthesized and secreted in a circadian manner with high levels occurring in all species at night. Melatonin has been implicated in the regulation of various neural and endocrine processes that are cued by the daily change in photoperiod (Masana and Dubocovich, 2001) Two mammalian melatonin receptor subtypes, MT₁ and MT₂ have been cloned. Both receptors are members of the superfamily of putative seven transmembrane G-protein coupled receptors, which are coupled to the inhibition of cyclic AMP synthesis upon agonist binding. Pharmacological studies suggest that the phase shifting effect of melatonin on circadian rhythms may be mediated by the MT₂ receptor subtype (Dubocovich et al. 1998). Melatonin inhibition of dopamine release from the retina is also mediated by the MT₂ receptor subtype (Dubocovich et al. 1997). Chemicon's MT₂ 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 agonists and antagonists at MT₂ . The membrane preparations exhibit a Kd of 0.1 nM for [¹²⁵ I]-Iodomelatonin. With 0.2 nM [¹²⁵ I]-Iodomelatonin, 5 µg/well MT₂ Membrane Prep typically yields greater than 6-fold signal-to-background ratio.

Radioligand binding assay and GTPγS binding

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Human

Table 1. Signal:background and specific binding values obtained in a competition binding assay with MT₂ membrane prep and unlabeled melatonin.

	5 µg/well
Signal:Background	9.96
Specific Binding (cpm)	1424.1

SPECIFICATIONS: 1 unit = 5 µg
Bmax for [¹²⁵ I]-Iodomelatonin binding: 0.04 pmol/mg protein
Kd for [¹²⁵ I]-Iodomelatonin binding: ~0.1 nM

Chem-1

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, 500mM NaCl. 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 , filtered and stored at 4°C
Radioligand: [¹²⁵ I] Iodomelatonin (Perkin Elmer#: NEX-236)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl, filtered and stored at 4°C.
One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 6-fold signal:background with [¹²⁵ I]-labeled Iodomelatonin at 0.2 nM

Chem-1, an adherent mammalian cell line lacking endogenous MT₂ expression.

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 1 mg/mL in packaging buffer, and dispensed at 1 mL/vial. Vials were rapidly frozen, and stored at -80°C.

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

• MTNR1B
• Mel-1B-R
• MT2
• MEL-1B-R

GPCR Membrane Preparations

GPCR