HTS075M
ChemiScreen Membrane Preparation
Recombinant Human M5 Muscarinic Acetylcholine Receptor
Human M5 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.
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About This Item
UNSPSC Code:
41106514
eCl@ss:
32161000
NACRES:
NA.84
Recommended Products
biological source
human
Quality Level
recombinant
expressed in Chem-1 cells
manufacturer/tradename
ChemiScreen
Chemicon®
technique(s)
ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable
NCBI accession no.
UniProt accession no.
shipped in
dry ice
Gene Information
human ... CHRM5(1133)
General description
Full-length human CHRM5 cDNA, encoding M5
The muscarinic acetylcholine receptor family consists of five GPCRs that mediate some of the neurotransmission functions of acetylcholine in the CNS and the periphery. The M5 receptor, along with the M1 and M3 receptors, signal through Gq/11 and subsequent release of Ca++ from the ER (Caulfield and Birdsall, 1998). Although M5 is expressed in the CNS at relatively low levels, it appears to be the only muscarinic acetylcholine receptor expressed in midbrain dopaminergic neurons, and it has been shown to mediate dopamine release from these neurons. The M5 receptor is also expressed in blood vessels in the brain and the periphery, and studies with M5 knockout mice demonstrate that M5 mediates the vasodilatory action of acetylcholine on cerebral microvessels. Further studies with M5 knockout mice also indicate a role for M5 in the rewarding effects of cocaine and morphine (Wess, 2004). Chemicon′s M5 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 M5 interactions with its ligands. The membrane preparations exhibit a Kd of 0.16 nM for [3H]-Scopolamine methyl chloride (NMS). With 10 μg/well M5 Membrane Prep and 1 nM [3H]-NMS, greater than 5-fold signal-to-background ratio was obtained.
Application
Radioligand binding assay and GTPγS binding.
Biochem/physiol Actions
GPCR Class: A
Protein Target: M5
Target Sub-Family: Acetylcholine (muscarinic)
Quality
Bmax: 2.49 pmol/mg; Kd: 0.16nM
1 unit = 10 μg
1 unit = 10 μg
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] NMS (Perkin Elmer#:NEX-636 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500 mM 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 5-fold signal:background with 3H-labeled NMS at 1 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, 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] NMS (Perkin Elmer#:NEX-636 )
Wash Buffer: 50 mM Hepes, pH 7.4, 500 mM 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 5-fold signal:background with 3H-labeled NMS at 1 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.
Storage Class Code
10 - Combustible liquids
WGK
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)
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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