ChemiSCREEN Human Y ₂ Neuropeptide Receptor Membrane Preparation

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of TP membrane prep:

	10 µg/well	5 µg/well
Signal:Background	19.8	26.3
Specific Binding (cpm)	27347	23335

SPECIFICATIONS: 1 unit = 5 µg
Bmax for [¹²⁵I] human PYY binding: 17.89 pmol/mg protein;
Kd for [¹²⁵I] human PYY binding: ~ 1.28 nM

Human Y2 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay and GTPγS binding.

GPCR Class: A

Protein Target: Y2

Target Sub-Family: Neuropeptide Y

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Inucbation 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, 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]-human PYY (Perkin Elmer#:NEX-341 )
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 a unit is the amount of membrane that will yield greater than 10-fold signal:background with ¹²⁵I labeled human PYY at 0.5 nM

Full-length human NPY2R cDNA encoding Y₂

The NPY family consists of three 36-amino acid peptides, neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), which bind to the NPY receptor family of G protein-coupled receptors. Five NPY receptors, Y₁, Y₂, Y₄, Y₅ and Y₆, have been defined at the molecular level, and each signals primarily through Gi/o. Binding of NPY family peptides to NPY receptors mediates a variety of physiological effects, including promotion of food intake, decreased anxiety, inhibition of neurotransmitter and hormone release, vasoconstriction, and gut motility. Y₂ is primarily expressed in the CNS, and it mediates presynaptic inhibition of neurotransmitter release (Michel et al., 1998). Chemicon′s Y₂ 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 Y₂ interactions and its ligands. The membrane preparations exhibit a Kd of 1.28 nM for [¹²⁵I]-human PYY. With 0.5 nM [¹²⁵I]-human PYY, 5 or 10µg/well Y₂ Membrane Prep yields greater than 10-fold signal-to-background ratio.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 0.5 mg/ml in 1 mL packaging buffer, rapidly frozen, and stored at -80°C.

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

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