Description
General description
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
Application
Radioligand binding assay, and GTPγS binding.
Biochem/physiol Actions
GPCR Class: A
Protein Target: BB1
Target Sub-Family: Bombesin
Features and Benefits
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.
Packaging
1 mL per vial
Physical form
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.
Preparation Note
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Analysis Note
Signal:background and specific binding values obtained in a competition binding assay with varying amounts of BB1 membrane prep:
10 µg/well5 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
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
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.
