M3 MEMBRANE PREPARATION

General description

Full-length human CHRM3 cDNA encoding M₃

The muscarinic acetylcholine receptor (mAChR) family consists of five GPCRs that mediate some of the neurotransmission functions of acetylcholine in the CNS and the periphery. The M₁, M₃ and M₅ receptors couple to G_q to mobilize intracellular calcium, whereas the M₂ and M₄ receptors couple to G_i/o to inhibit cAMP production (Caulfield and Birdsall, 1998). M₃ is expressed prominently in smooth muscle, and plays a primary role in mediating mAChR agonist-induced contractility. Mice lacking M₃ have dilated pupils, which indicates a role for M₃ in regulating tone of the pupillary sphincter muscle. In addition, Msub3 plays a role in feeding, as indicated by the lean and hypophagic phenotype of M₃-null mice (Wess, 2004). Chemicon′s M₃ 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 M₃ interactions with 4-DAMP. The membrane preparations exhibit a Kd of 0.72-1 nM for [³H]-4-DAMP. With 10 µg/well M₃ Membrane Prep and 0.75 nM [³H]-4-DAMP, a greater than 4-fold signal-to-background ratio was obtained.

Application

Human M3 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

Radioligand binding assay and GTPγS binding.

Biochem/physiol Actions

GPCR Class: A

Protein Target: M3

Target Sub-Family: Acetylcholine (muscarinic)

Quality

Signal:background and specific binding values obtained in a competition binding assay with varying amounts of M₃ membrane prep:

Specifications

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: [³H]-4-DAMP (Perkin Elmer#:NET1040 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM 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 4-DAMP at 0.75 nM

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.

Storage and Stability

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

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.

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