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Figure 1. Saturation binding for M₂. 10 µg/well M₂ Membrane Preparation was incubated with increasing amount of ³. ...

Figure 2. Competition binding for M₂. 10 µg/well M₂ Membrane Preparation (HTS115M) was incubated with 0.5 nM ³H-lab ...

ChemiSCREEN™ Membrane Preparation Recombinant Human M₂ Muscarinic Acetylcholine Receptor

Description:

Trade Name:

ChemiScreen

Product Overview:

Full-length human CHRM2 cDNA encoding M₂

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 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). In urinary bladder trachea and stomach, M₂ augments the function of M₃ in promoting contractility, and activation of M₂ serves to counteract relaxation induced by increased cAMP levels (Ehlert et al., 2005; Wess, 2004). In addition, the ability of mAChR agonists to decrease heart rate appears to be mediated primarily by M₂. Agonists of mAChRs induce tremor, hypothermia, corticosterone release, and analgesia; each of these functions is mediated at least in part by M₂ (Wess, 2004). Chemicon's M₂ 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 M₂ interactions with its ligands. The membrane preparations exhibit a Kd of 0.24 nM for [³H]-Scopolamine methyl chloride (NMS). With 10 µg/well M₂ Membrane Prep and 0.5 nM [³H]-NMS, greater than 3-fold signal-to-background ratio was obtained.

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.

Alternate Names:

mAChR2

Host Cells:

Chem-1, an adherent mammalian cell line without any endogenous M₂ expression.

Cell Type:

Chem-1

Gene Symbol:

CHRM2
MGC120006
FLJ43243
MGC120007
HM2

Presentation:

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

Quality Assurance:

Table 1. Signal:background and specific binding values obtained in a competition binding assay with M₂ membrane prep and unlabeled Atropine.

	10 µg/well
Signal:Background	3.2
Specific Binding (cpm)	606.8

SPECIFICATIONS: 1 unit = 10 µg
Bmax for [³H]-NMS binding: 0.5 pmol/mg protein
K_d for [³H]-NMS binding: ~0.24 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, an FC 96-well harvest plate (Millipore cat. # MAHF C1H) is coated with 0.33% polyethyleneimine for 30 min, then washed with 4mM Na₂HPO₄, 1mM KH₂PO₄, pH7.4. 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 and Wash buffer: 4mM Na₂HPO₄, 1mM KH₂PO₄, pH7.4, filtered and stored at 4°C Radioligand: [³H] NMS (Perkin Elmer#:NEX-636 ) One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 3-fold signal:background with ³H-labeled NMS at 0.5 nM

Storage Conditions:

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

Species:

Human