Anti-EAAT2 (extracellular) Antibody

Excitatory Amino Acid Transporter 2, GLT-1, SLC1A2
    Cat #: AGC-022
  • Lyophilized Powder
  • Antigen Incl.
  • Shipped at Room Temp.
  • Type: Polyclonal
    Source: Rabbit
    Reactivity: h, m, r
    Immunogen
    Peptide (C)KQLGPGKKNDEVS, corresponding to amino acid residues 151-163 of rat EAAT2 (Accession P31596).  2nd extracellular loop.
    Accession (Uniprot) Number P31596
    Gene ID 29482
    Peptide confirmation Confirmed by amino acid analysis and mass spectrometry.
    Homology Mouse, human - identical.
    Purity Affinity purified on immobilized antigen.
    Formulation Lyophilized powder. Reconstituted antibody contains phosphate buffered saline (PBS), pH 7.4, 1% BSA, 0.025% NaN3.
    Storage before reconstitution The antibody ships as a lyophilized powder at room temperature. Upon arrival, it should be stored at -20°C.
    Reconstitution 25 µl, 50 μl or 0.2 ml double distilled water (DDW), depending on the sample size.
    Antibody concentration after reconstitution 0.8 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, lci, wb
    May also work in: ifc, ip
    Western blot
    Western blot analysis of rat cerebellum lysate:
    1.  Anti-EAAT2 (extracellular) Antibody (#AGC-022), (1:500).
    2.  Anti-EAAT2 (extracellular) Antibody, preincubated with the control peptide antigen.
    Western blot analysis of mouse brain membranes:
    1.  Anti-EAAT2 (extracellular) Antibody (#AGC-022), (1:200).
    2.  Anti-EAAT2 (extracellular) Antibody, preincubated with the control peptide antigen.
    Immunohistochemistry
    Expression of EAAT2 in rat hippocampus
    Immunohistochemical staining of perfusion-fixed, free floating frozen rat brain sections with Anti-EAAT2 (extracellular) Antibody (#AGC-022). In the rat hippocampal CA1 region EAAT2 immunoreactivity (green) appears in the neuropil in the stratum oriens (S. Or) and in the pyramidal layer (Pyr) and was particularly intense around pyramidal neurons (arrow in A and compare with DAPI counterstain in B).
    Live cell imaging / Immunocytochemistry
    Expression of EAAT2 in human U-87 MG cells
    Immunocytochemical staining of live intact human U-87 MG glioblastoma cells. A. Extracellular staining of cells using Anti-EAAT2 (extracellular) Antibody (#AGC-022), (1:100) followed by goat anti-rabbit-Alexa-Fluor-594 secondary antibody (red). B. Live image of the cells. C. Merge of A and B.
    References
    1. Tzingounis, A.V. and Wadiche, J.I. (2007) Nat. Rev. Neurosci. 8, 935.
    2. Beart, P.M. and O’Shea, R.D. (2007) Br. J. Pharmacol. 150, 5.
    Scientific background

    L-Glutamate (Glu) is an abundant amino acid that functions as the major excitatory neurotransmitter in the central nervous system. However, excess of Glu in the extracellular synaptic milieu leads to neuronal cell death by a process known as excitotoxicity.

    The extracellular levels of Glu are regulated by a family of high affinity plasma membrane transporters called excitatory amino acid transporters (EAATs) which are responsible for the re-uptake of Glu into the cells1,2.

    The EAAT family includes five members (EAAT1-EAAT5) that are members of the solute carrier family 1 (SLC1) of Na+-dependent transporters that also includes the neutral amino acid transporters ASCT1 and ASCT2.

    The Glu transporters present an unusual topology of eight transmembrane domains with two re-entrant loops and intracellular N- and C- termini. The transporter is likely assembled as a trimer where each monomer is a functional unit capable of binding the Glu substrate.

    The transport of Glu into the cells by the EAAT transporters is coupled to the Na+ and K+ electrochemical gradient as a driving force. Hence, the uptake of Glu is dependent on the co-transport of three Na+ and one H+ ions, and the counter transport of one K+ ion. 

    In addition, to the well documented Glu uptake, the EAAT transporters show a Glu-independent Cl- conductance. The physiological significance of the Cl- current through the EAATs is currently unknown1,2.

    EAAT2 (also known as Glutamate Transporter-1, GLT-1) as well as EAAT1, is expressed predominantly in glia cells, while EAAT3EAAT4 and EAAT5 are mostly expressed in neurons.

    As mentioned earlier, EAAT transporters represent the only (significant) mechanism for removal of glutamate from the extracellular fluid and hence are essential for the long-term maintenance of low and non-toxic concentrations of glutamate and the preservation of normal excitatory synaptic transmission.

    In addition to Glu uptake, the glutamate transporters provide glutamate for the synthesis of γ-Aminobutyric acid (GABA), glutathione and protein, suggesting an interactive role between EAATs and cellular metabolism1,2.

    Dysregulation of EAAT activities has been implicated in several neurodegenerative disorders such as Alzheimer’s disease, traumatic brain injury, epilepsy and schizophrenia, suggesting that EAATs can be a useful target for the treatment of these conditions1,2.

    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
    Last update: 21/11/2018

    Anti-EAAT2 (extracellular) Antibody (#AGC-022) is a highly specific antibody directed against an epitope located at the second extracellular loop of the rat Excitatory Amino Acid Transporter 2. The antibody can be used in western blot, live cell imaging, and immunohistochemistry applications. It has been designed to recognize EAAT2 from human, rat, and mouse samples.

    For research purposes only, not for human use