Anti-TrkB (extracellular)-ATTO-488 Antibody

Neurotrophic tyrosine kinase receptor type 2, NTRK2, BDNF/NT-3 growth factors receptor
    Cat #: ANT-019-AG
  • Lyophilized Powder
  • Antigen Incl.
  • Shipped at Room Temp.
  • Type: Polyclonal
    Source: Rabbit
    Reactivity: h, m, r
    Immunogen
    Peptide (C)RLEPNSIDPENITE, corresponding to amino acid residues 57-70 of rat TrkB (Accession Q63604). Extracellular domain.
    Accession (Uniprot) Number Q63604
    Gene ID 25054
    Peptide confirmation Confirmed by amino acid analysis and mass spectrometry.
    Homology Mouse, human - 13/14 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, 0.025% NaN3.
    Label ATTO-488. Maximum absorption 501 nm; maximum fluorescence 523 nm. The fluorescence is excited most efficiently in the 480 – 515 nm range. This label is analogous to the well known dye fluorescein isothiocyanate (FITC) and can be used with filters typically used to detect FITC.
    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 double distilled water (DDW).
    Antibody concentration after reconstitution 1 mg/ml.
    Storage after reconstitution The reconstituted solution can be stored at 4°C, protected from the light, 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 × 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 -20°C.
    Preadsorption Control 1 µg peptide per 1 µg antibody.
    Standard quality control of each lot Western blot analysis (unlabeled antibody, #ANT-019), and immunohistochemistry (labeled antibody).
    Applications: fc, ih
    May also work in: ic, lci
    Immunohistochemistry
    Expression of TrkB in rat DRG
    Immunohistochemical staining of rat dorsal root ganglia (DRG) frozen sections using Anti-TrkB (extracellular)-ATTO-488 Antibody (#ANT-019-AG), (1:50), (green). Staining is present in DRG neurons. Hoechst 33342 is used as the counterstain (blue).
    BDNF co-localizes with TrkB in mouse cerebellum region.
    A. Fixed brain sections were incubated with human BDNF-Biotin (#B-250-B) followed by Streptavidin-Cy3. B. Same sections were incubated with Anti-TrkB (extracellular)-ATTO-488 Antibody (#ANT-019-AG). C. Merge of A and B reveals co-localization in the Purkinje layer (P). TrkB positive cells (green) with BDNF binding (red, arrow) is seen.
    Immuno-colocalization of Aquaporin 4 and TrkB in rat cerebellum.
    Immunohistochemical staining of perfusion-fixed frozen rat cerebellum sections using Anti-TrkB (extracellular)-ATTO-488 Antibody (#ANT-019-AG), (1:80) and Anti-Aquaporin 4 (300-314)-ATTO-594 Antibody (#AQP-014-AR), (1:80). A. TrkB labeling (green) appears in Purkinje cells (arrows) forming the Purkinje layer (P). B. Aquaporin 4 staining (red) appears in outlines of the “baskets” formed around Purkinje cells (vertical arrows) and in some blood vessels (horizontal arrow). C. Merge of panels A and B demonstrates the localization of TrkB and Aquaporin 4 in adjacent cellular compartments of the cerebellum. Nuclei were demonstrated using DAPI as the counterstain (blue).
    Direct flow cytometry
    10 µg antibody/1x106 human HL-60 promyelocytic leukemia cells.
    References
    1. Webster, N.J.G. and Pirrung, M.C. (2008) BMC Neurosci. 9, S1.
    2. Quartu, M. et al. (2003) Int. J. Dev. Neurosci. 21, 309.
    3. Aoki, C. et al. (2000) J. Neurosci. Res. 59, 454.
    4. Yan, Q. et al. (1997) J. Comp. Neurol. 378, 135.
    5. Patapoutian, A. and Reichardt, L.F. (2001) Curr. Opin. Neurobiol. 11, 272.
    6. Schecterson, L.C. and Bothwell, M. (2010) Develop. Neurobiol. 70, 332.
    7. Grimes, M.L. et al. (1996) J. Neurosci. 16, 7950.
    Scientific background

    BDNF and NT-4 belong to the neurotrophin family which also includes NGF and NT-3. These neurotrophins bind two groups of receptors. The p75NTR receptor is common to all four neurotrophins and is a member of the tumor necrosis factor receptor family. The tropomyosin-related kinase (Trk) receptors are receptor tyrosine kinases (RTKs) and three receptors form this family: TrkA, TrkB, and TrkC1.

    As mentioned above, the p75NTR receptor binds to all neurotrophins with similar affinities while the Trk receptors are the ones to display the selectivity for the neurotrophins. TrkA is activated by NGF binding, TrkB by that of BDNF and NT-4, while TrkC is stimulated by the binding of NT-31.

    All three Trk receptors are highly expressed in the mammalian brain in very distinct regions and are also expressed in the peripheral nervous system2-4. Cholinergic neurons in the basal forebrain exclusively express TrkA, while TrkB and TrkC are highly expressed in the hippocampus. Motor and sensory neurons in the peripheral nervous system express Trk receptors. Interestingly, Trk receptors are not essential for development, but knockout mice die shortly after birth. Indeed, TrkB-deficient mice demonstrate a significant decrease in motor neurons and synaptogenesis1.

    Trk receptors have many motifs in the extracellular region, including cell-adhesion domains, three tandem leucine rich motifs flanked by two clusters of cysteines. In the membrane proximal region of the receptor there are also two immunoglobulin-like domains5. The binding of neurotrophins to Trk receptors promotes receptor dimerization resulting in kinase activation. Activated Trk receptors then phosphorylate a cascade of signaling molecules including the Ras/ERK, PI3K/Akt pathways and PLC-g1. Activated Trk receptors also create internal docking sites for other signaling adaptor proteins to bind to5. Splice variants of TrkA, TrkB and TrkC have been observed. These splice isoforms are mainly affected in the tyrosine kinase domain of the receptor lying in the cytoplasm. Endocytosis is an important signaling trait of Trk receptors.

    Following neurotrophin binding to the Trk receptor, the receptor complex is then internalized via endocytosis in order to terminate signaling. However, in the axonal compartment of neurons the internalization process of the neurotrophin complexed to the receptor is part of the signaling process and is important for activating transcription processes in the nucleus6,7.

    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:

    Anti-TrkB (extracellular)-ATTO-488 Antibody
    BDNF co-localizes with TrkB in mouse hippocampal CA1 region.A. Fixed brain sections were incubated with human BDNF-Biotin (#B-250-B) followed by Streptavidin-Cy3. B. Same sections were incubated with Anti-TrkB (extracellular)-ATTO-488 Antibody (#ANT-019-AG). C. Merge of A and B reveals co-localization in the pyramidal layer (P). Outside the pyramidal layer, there are also cells with only BDNF (red, horizontal arrows) or only TrkB expression (green, vertical arrows).

    Last update: 23/10/2018

    Anti-TrkB (extracellular) Antibody (#ANT-019) is a highly specific antibody directed against an epitope of the rat protein. The antibody can be used in western blot, immunohistochemistry, indirect flow cytometry, and live cell imaging applications. It has been designed to recognize TrkB from rat, mouse, and human samples.

    Anti-TrkB (extracellular)-ATTO-488 Antibody (#ANT-019-AG) is directly labeled with an ATTO-488 fluorescent dye. ATTO dyes are characterized by strong absorption (high extinction coefficient), high fluorescence quantum yield, and high photo-stability. The ATTO-488 label is analogous to the well known dye fluorescein isothiocyanate (FITC) and can be used with filters typically used to detect FITC. Anti-TrkB (extracellular)-ATTO-488 Antibody is specially suited to experiments requiring simultaneous labeling of different markers.

    For research purposes only, not for human use
    Citations
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