ChemiSCREEN CB₁ Membrane Preparation

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

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

Radioligand binding assay, and GTPγS binding.

GPCR Class: A

Protein Target: CB1

Target Sub-Family: Cannabinoid

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

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. 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

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.

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

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