Description
General description
Bradykinins bind and activate two GPCRs, termed B1 and B2 (gene names BDKRB1 and BDKRB2, respectively), which signal through Gq and Gi. B2 is widely expressed in a constitutive fashion, whereas B1 expression is induced during inflammation. B2 appears to mediate much of the activity of kinins in vasodilation, arthritis, edema, and nociception (Leeb-Lundberg et al., 2005). Chemicon′s B2 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 B2 interactions with bradykinin. The membrane preparations exhibit a Kd of 0.18 nM for [3H]-bradykinin. With 5.0 μg/well, B2 Membrane Prep and 0.4 nM [3H]-bradykinin, a greater than 4-fold signal-to-background ratio is obtained.
Full length Human BDKRB2
Application
Radioligand binding assay, and GTPgammaS binding
Biochem/physiol Actions
GPCR Class: A
Protein Target: B2
Target Sub-Family: Bradykinin
Features and Benefits
Inucbation Conditions
RECOMMENDED ASSAY 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: [3H] Bradykinin, [2,3-prolyl-3,4-3H(N)] (Perkin Elmer #NET-706)
Wash Buffer: 50 mM Hepes, pH 7.4, 500 mM 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 4-fold signal:background with 3H-labeled bradykinin at 0.4 nM
Physical form
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
SPECIFICATIONS: 1 unit = 10 µg
Bmax: 2.73 pmol/mg
Kd: 0.18 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.
