ChemiSCREEN Membrane Preparation Recombinant Human NPBW₂ Neuropeptide B/W Receptor

FUNCTION: SwissProt: P48146 # Interacts specifically with a number of opioid ligands. Receptor for neuropeptides B and W, which may be involved in neuroendocrine system regulation, food intake and the organization of other signals.
SIZE: 333 amino acids; 36861 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Detected at high levels in caudate nucleus, hyppocampus and amygdala; at moderate levels in the adult brain, thalamus, parietal cortex, pituitary gland, adrenal gland and lymph nodes.
SIMILARITY: SwissProt: P48146 ## Belongs to the G-protein coupled receptor 1 family.

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 was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

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

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 Tris, pH 7.4. 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: [¹²⁵I]-neuropeptide W23 (Perkin Elmer#: NET-403)
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 an unit is the amount of membrane that will yield greater than 10-fold signal:background with ¹²⁵I-labeled neuropeptide W23 at 0.35nM.

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