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Figure 1. Saturation binding for M₃. 10 µg/well M₃ Membrane Preparation was incubated with increasing amount of [^{3 ...}

Figure 2. Competition binding for M₃. M₃ Membrane Preparation (20 or 10 µg/well) or Wild-Type Chem-1 membrane preparati ...

ChemiSCREEN™ Human M₃ Muscarinic Acetylcholine Receptor Membrane Preparation

Description:

Trade Name:

ChemiScreen

Product Overview:

Full-length human CHRM3 cDNA encoding M₃

Background Information:

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 Gq to mobilize intracellular calcium, whereas the M2 and M4 receptors couple to Gi/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, Ms<LB/>ub<RB/>3 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 Notes:

Radioligand binding assay and GTPγS binding.

Species Reactivity:

Human

Usage Statement:

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.

Host Cells:

Chem-1, an adherent mammalian cell line with no endogenous M₃ expression.

Cell Type:

Chem-1

Gene Symbol:

CHRM3
HM3

Presentation:

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 Assurance:

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

	20 µg/well	10 µg/well
Signal:Background	5.17	5.10
Specific Binding (cpm)	1224	1030

SPECIFICATIONS: 1 unit = 10 µg
Bmax: 3.75 pmol/mg
Kd: 0.86 nM

Protein or Enzyme Type:

Membrane Preparations

Incubation 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

Storage Conditions:

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

Species:

Human