ChemiScreen Recombinant Human BB1 Bombesin/Neuromedin B Receptor Membrane Preparation

Bombesin, a bioactive peptide first identified in amphibian skin, is related to two mammalian peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB). A family of 3 GPCRs, including NMB-R (BB₁), GRP-R (BB₂), and BRS-3 (BB₃), mediate the biological effects of the peptides. The receptors differ in their affinities for the peptides; BB2 binds to GRP with 50-300-fold greater affinity than to NMB, whereas BB1 binds to NMB with 10-800-fold greater affinity than to GRP (Tokita et al., 2004). Binding of ligand to BB1 activates Gq to increase intracellular calcium concentrations. The CNS is a major site of NMB and BB1 expression, and BB₁ appears to be involved in thermoregulation (Ohki-Hamazaki et al., 2005). Chemicon′s BB₁ 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 BB1 interactions with neuromedin B. The membrane preparations exhibit a Kd of 0.35-0.43 nM for [¹²⁵I]-Bombesin. With 5 µg/well BB₁ Membrane Prep and 0.3 nM [¹²⁵I]-bombesin, a greater than 40-fold signal-to-background ratio was obtained.

Full-length human NMBR cDNA encoding BB1

Radioligand binding assay, and GTPγS binding.

GPCR Class: A

Protein Target: BB1

Target Sub-Family: Bombesin

1 mL per vial

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of BB₁ membrane prep:

	10 µg/well	5 µg/well
Signal:Background	82.5	74.8
Specific Binding (cpm)	27377	22443

SPECIFICATIONS: 1 unit = 5 µg membrane preparation
Bmax: 3.1 pmol/mg
Kd: 0.39 nM

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] bombesin (Perkin Elmer # NEX258)
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 an unit is the amount of membrane that will yield greater than 40-fold signal:background with ¹²⁵I-labeled bombesin at 0.3 nM.

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 the indicated concentration in 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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