ChemiSCREEN Membrane Preparation Recombinant Human BB ₃ Bombesin Receptor

32161000

Full length human BRS3 encoding BB₃
Bombesin, a bioactive peptide first identified in amphibian skin, is related to two mammalian peptides, gastrin-releasing peptide (GRP) and neuromedin B. A family of 3 GPCRs, including GRP-R (BB₁), NMB-R (BB₂) and BRS-3 (BB₃), mediate the biological effects of the peptides (Ohki-Hamazaki et al., 2005). BB₃ differs from the others by its low affinity for bombesin. Although an endogenous ligand for BB₃ has yet to be identified, a synthetic nonselective bombesin-like peptide [H-D-Phe6, β-Ala11, Phe13, Nle14]-bombesin-(6-14)-nonapeptide amide (Bombesin (6-14) Analog) activates BB₃ with high potency. BB₃-null mice have an obese phenotype (Matsumoto and Iijima, 2003). Millipore′s BB₃ 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 BB₃ receptor interactions with its ligand. The membrane preparations exhibit a Kd of 0.23nM for [125I]-Bombesin (6-14) Analog. With 10 μg/well BB₃ Membrane Prep and 0.3 nM [¹²⁵I]-Bombesin (6-14) Analog, a greater than 3-fold signal-to-background ratio was obtained.

Human BB3 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.
Radioligand binding assay and GTPγS binding

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 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, 0.2 mg/ml bacitracin, 20 μg/ml leupeptin, 20 μg/ml chymostatin, 1 Protease Inhibitor cocktail Tablets (Roche Cat. No. 11 873 580 001) for each 50 ml binding buffer.

Radioligand: [¹²⁵I]-Bombesin (6-14) Analog (Perkin Elmer NEX377)

Wash Buffer: 50 mM Hepes, pH 7.4, 500 mM NaCl , 0.1% BSA, filtered and stored at 4℃.

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 3-fold signal:background with ¹²⁵I-labeled Bombesin (6-14) Analog 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 was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

100

human

expressed in Chem-1 cells

ChemiScreen
Chemicon^®

ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable

NM_001727

P32247

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

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

12 - Non Combustible Liquids

WGK 2