HTS115M
ChemiSCREEN Membrane Preparation Recombinant Human M2 Muscarinic Acetylcholine Receptor
Human M2 GPCR membrane prepation for Radioligand binding Assays.
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About This Item
UNSPSC Code:
41106514
eCl@ss:
32161000
NACRES:
NA.41
biological source
human
Quality Level
recombinant
expressed in Chem-1 cells
manufacturer/tradename
ChemiScreen
Chemicon®
technique(s)
radioligand binding assay (RLBA): suitable
NCBI accession no.
UniProt accession no.
shipped in
dry ice
Gene Information
human ... CHRM2(1129)
General description
Full-length human CHRM2 cDNA encoding M2
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.
Biochem/physiol Actions
Protein Target: M2
Quality
Table 1. Signal:background and specific binding values obtained in a competition binding assay with M2 membrane prep and unlabeled Atropine.
SPECIFICATIONS: 1 unit = 10 µg
Bmax for [3H]-NMS binding: 0.5 pmol/mg protein
Kd for [3H]-NMS binding: ~0.24 nM
| 10 µg/well | |
|---|---|
| Signal:Background | 3.2 |
| Specific Binding (cpm) | 606.8 |
Bmax for [3H]-NMS binding: 0.5 pmol/mg protein
Kd for [3H]-NMS binding: ~0.24 nM
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, 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
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
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.
Packaging method: Membranes protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80oC.
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Storage Class
12 - Non Combustible Liquids
wgk_germany
WGK 2
Certificates of Analysis (COA)
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Find documentation for the products that you have recently purchased in the Document Library.
International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors.
M P Caulfield et al.
Pharmacological reviews, 50(2), 279-290 (1998-07-02)
Frederick J Ehlert et al.
The Journal of pharmacology and experimental therapeutics, 313(1), 368-378 (2004-12-21)
We investigated the contractile role of M2 muscarinic receptors in mouse urinary bladder. When measured in the absence of other agents, contractions elicited to the muscarinic agonist oxotremorine-M exhibited properties consistent with that expected for an M3 response in urinary
Muscarinic acetylcholine receptor knockout mice: novel phenotypes and clinical implications.
Wess, Jurgen
Annual Review of Pharmacology and Toxicology, 44, 423-450 (2004)
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