CB1 MEMBRANE PREPARATION-200U

Application

Radioligand binding assay, and GTPγS binding.

Human CB1 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Biochem/physiol Actions

Target Sub-Family: Cannabinoid

GPCR Class: A

Protein Target: CB1

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.

General description

CB₁ is a GPCR that is expressed primarily in brain and nervous tissue, and mediates numerous CNS responses such as analgesia, appetite, cognition, memory and locomotor activity. A number of cannabinoid ligands bind to CB₁ and activate Gi/o-mediated downstream responses, including inhibition of cAMP production and activation of ion channels and MAP kinases. Ligands for CB₁ include exogenous agonists such as Delta9-THC, the main psychoactive component of the plant Cannabis sativa, and endogenous eicosanoid agonists such as anandamide. A number of synthetic agonists such as CP55940 and R-(+)-WIN55212, and antagonists, such as SR141716A, for CB₁ have been developed (Howlett et al., 2002). CB₁ agonists have clinical utility in analgesia and antiemetic properties, whereas CB₁ antagonists show promise for treatment of appetite in obesity disorders. Chemicon′s CB₁ 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 for agonists and antagonists at CB1. The membrane preparations exhibit a Kd of 7 nM for [³H]-SR141716A. In the presence of 2 nM [³H]-SR141716A with CP55940 as unlabeled competitor, 5 µg/well and 10 µg/well CB₁ Membrane Prep yield 4- and 5-fold signal-to-background ratios, respectively.

Human CB₁

Legal Information

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

Physical form

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.Packaging method: Membranes protein were adjusted to 0.5 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C.

Quality

SPECIFICATIONS:
1 unit = 10 µg
Bmax: 15 pmol/mg
K_d: 7 nM for SR141716A

Specifications

Inucbation ConditionsRECOMMENDED 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. Ligands were diluted in binding buffer containing 30% DMSO, then added to membranes such that the final DMSO concentration was 15%.Radioligand: [³H] SR141716A (Amersham #TRK1028)

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

One vial contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield approximately 4-fold signal:background with [³H] SR141716A at 2 nM

Storage and Stability

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