ChemiSCREEN Human Y ₂ Neuropeptide Receptor Membrane Preparation

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.

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

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.

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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