M2 Membrane Preparation

Code: HTS115M D2-231

Biochem/physiol Actions

Protein Target: M2

General description

The muscarinic acetylcholine receptor (mAChR) family consists of five GPCRs that m...


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Biochem/physiol Actions

Protein Target: M2

General description

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). 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.

Full-length human CHRM2 cDNA encoding M2

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 was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.

Quality

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
Kd for [3H]-NMS binding: ~0.24 nM

Specifications

Inucbation ConditionsMembranes 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

biological sourcehuman
Gene Informationhuman ... CHRM2(1129)
manufacturer/tradenameChemicon®, ChemiScreen
NCBI accession no.NM_001006626.1, NM_001006628.1, NM_001006631.1, NM_001006627.1, NM_000739.2, NM_001006629.1, NM_001006630.1, NM_001006632.1
Quality Level100
recombinantexpressed in Chem-1 cells
shipped indry ice
technique(s)radioligand binding assay (RLBA): suitable
UniProt accession no.P08172
This product has met the following criteria to qualify for the following awards:



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