Anti-M4 Muscarinic Receptor Antibody

Muscarinic acetylcholine receptor M4, Chrm4
    Cat #: AMR-004
  • Lyophilized Powder
  • Antigen Incl.
  • Shipped at Room Temp.
  • Type: Polyclonal
    Source: Rabbit
    Reactivity: h, m, r
    Immunogen
    Peptide(C) RTLNPASKWSK, corresponding to amino acid residues 330-340 of rat M4 Muscarinic Receptor (Accession  P08485). 3rd intracellular loop.
    Accession (Uniprot) Number P08485
    Gene ID 25111
    Peptide confirmation Confirmed by amino acid analysis and mass spectrometry.
    Homology Mouse - identical; human - 9/12 amino acid residues identical.
    Purity Affinity purified on immobilized antigen.
    Formulation Lyophilized powder. Reconstituted antibody contains phosphate buffered saline (PBS), pH 7.4, 1% BSA, 0.05% NaN3.
    Storage before reconstitution The antibody ships as a lyophilized powder at room temperature. Upon arrival, it should be stored at -20°C.
    Reconstitution 50 µl or 0.2 ml double distilled water (DDW), depending on the sample size.
    Antibody concentration after reconstitution 0.85 mg/ml.
    Storage after reconstitution The reconstituted solution can be stored at 4°C for up to 1 week. For longer periods, small aliquots should be stored at -20°C. Avoid multiple freezing and thawing. Centrifuge all antibody preparations before use (10000 x g 5 min).
    Control antigen storage before reconstitution Lyophilized powder can be stored intact at room temperature for 2 weeks. For longer periods, it should be stored at -20°C.
    Control antigen reconstitution 100 µl double distilled water (DDW).
    Control antigen storage after reconstitution -20ºC.
    Preadsorption Control 1 µg peptide per 1 µg antibody.
    Standard quality control of each lot Western blot analysis.
    Applications: ic, ih, wb
    May also work in: ifc, ip
    Western blot
    Western blot analysis of rat brain membrane (lanes 1 and 2), mouse brain membrane (lane 3 and 5) and SH-SY5Y cell lystae (lanes 4 and 6):
    1, 3, 4. Anti-M4 Muscarinic Receptor Antibody (#AMR-004), (1:200).
    2, 5, 6. Anti-M4 Muscarinic Receptor Antibody, preincubated with the control peptide antigen.
    Immunohistochemistry
    Expression of M4 Muscarinic Receptor in mouse islands of Calleja
    Immunohistochemical staining of mouse islands of Calleja (ventral striatum) using Anti-M4 Muscarinic Receptor Antibody (#AMR-004).  A. M4 Muscarinic Receptor staining (red) appears in soma of neuronal profiles (vertical arrows) and a diffuse halo (horizontal arrows) in the vicinity of the islands of Calleja (IC).  B. Nuclear staining using DAPI as the counterstain. C. Merged image of A and B.
    Immunocytochemistry
    Expression of M4 muscarinic receptor in C6 transfected cells
    Immunocytochemical staining of paraformaldehyde-fixed and permeabilized C6 cells. A. Transfected cells were stained using Anti-M4 Muscarinic Receptor Antibody (#AMR-004), (1:100) followed by goat anti-rabbit-AlexaFluor-594 secondary antibody (red). B. Non-transfected cells were stained using Anti-M4 Muscarinic Receptor antibody (1:100), followed by goat anti-rabbit-AlexaFluor-594 secondary antibody (red). DAPI is used as the counterstain (blue).
    References
    1. Nathanson, N.M. (2008) Pharmacol. Ther. 119, 33.
    2. Migeon, J.C. and Nathanson N.M. (1994) J. Biol. Chem. 269, 9767.
    3. Ashkenazi, A. et al. (1987) Science 238, 672.
    4. Tietje, K.M. et al. (1990) J. Biol. Chem. 265, 2828.
    5. Katz, A. et al. (1992) Nature 360, 686.
    6. Perez-Burgos, A. et al. (2010) Neuroscience 165, 293.
    7. Dorofeeva, N.A. et al. (2009) Neuroreport 20, 1386.
    Scientific background

    Acetylcholine is a neurotransmitter which activates two different groups of receptors: nicotinic acetylcholine receptors (nAChRs) which belong to the superfamily of ligand-gated ion channels and muscarinic acetylcholine receptors (mAChR), belonging to the G-protein coupled receptor (GPCR) superfamily. There are five separate gene products that put together this GPCR subfamily, M1-M5. Generally, M1M3 and M5 muscarinic receptors are known to activate phospholipase C (PLC) via Gq coupling, while M2 and M4 muscarinic receptors couple to Gi/o, and therefore inhibit adenylate cyclase1. This classification is however not clear cut. mAChR can activate adenylate cyclase by coupling to Gs2. In addition, M2 and M4 muscarinic receptors, when overexpressed can also activate adenylate cyclase in some systems3-5.

    Evidence suggests that muscarinic receptors can form homo or heterodimers at that the dimer formed can subsequently affect the downstream signaling pathways1.

    Muscarinic receptors have been shown to regulate voltage-gated Ca2+ channels, namely Cav2.1 6 as well acid sensing ion channels (ASIC)7. Generally speaking the actions of muscarinic receptors on ion channels can be either via 2nd messengers or through their direct action on the channels once activated8.

    Furthermore, muscarinic receptors can also promote endocytosis of ion channels (KV1.2 for example) by recruiting tyrosine kinases that phosphorylate the channel in order to terminate its activity9. Other diverse and important functions of muscarinic receptors include cell growth, survival and physiology1.

    Expression of muscarinic receptors is found in neurons in the central nervous system as well as in the peripheral nervous system. In the non-nervous system, these receptors are expressed in the cardiac and smooth muscle, lung, intestine, ovary and urothelium1,10. M4 muscarinic receptor is mostly expressed in the striatum where it colocalizes with D1 dopamine receptors11-13.

    Malfunction of M4 muscarinic receptor is associated with a number of psychological and neurodegenerative diseases like schizophrenia, Alzheimer’s and Parkinson’s diseases11,14,15.

    Application key:

    CBE- Cell-based ELISA, FC- Flow cytometry, ICC- Immunocytochemistry, IE- Indirect ELISA, IFC- Indirect flow cytometry, IHC- Immunohistochemistry, IP- Immunoprecipitation, LCI- Live cell imaging, N- Neutralization, WB- Western blot

    Species reactivity key:

    H- Human, M- Mouse, R- Rat
    Image & Title:
    Immuno-colocalization of GluN1 and M4 Muscarinic Receptor in mouse parietal cortexImmunohistochemical staining of immersion-fixed, free floating mouse brain frozen sections using rabbit Anti-M4 Muscarinic Receptor Antibody (#AMR-004), (1:400) and Guinea pig Anti-NMDAR1 (GluN1) (extracellular) Antibody (#AGP-046), (1:1200). A. M4 Muscarinic receptor staining (green). B. GluN1 staining (red) in same section. C. Merge of the two images reveals several cells expressing both M4 Muscarinic Receptor and GluN1 (arrows). Cell nuclei were stained with DAPI (blue).
    Last update: 01/11/2018

    Anti-M4 Muscarinic Receptor Antibody (#AMR-004) is a highly specific antibody directed against an epitope of the rat protein. The antibody can be used in western blot, immunocytochemistry, and immunohistochemistry applications. It has been designed to recognize M4 muscarinic receptor from rat, mouse, and human samples.

    For research purposes only, not for human use