Our Anti-TrkB, phospho Tyr816/Y817 (TrkB, pY816/817) rabbit polyclonal primary antibody detects chicken (predicted), human, mouse, and rat (predicted) TrkB, phospho Tyr816/Y817 (TrkB, pY816/817), and is affinity-purified. It is validated for use in WB.
Western blot analysis of TrkB pY817 expression in NSC34 RIPA cell lysate (10 μg/lane) and mouse cortex homogenate (30 μg/lane). NSC34 cells were grown in proliferation medium in presence and absence of Retinoic Acid (RA, 1 μM) for 5 days. Cells were then serum-starved for 4 hours and treated with 50 ng/mL BDNF for 30 min. SDS-PAGE: 4-12%, reducing conditions; Transfer: Towbin’s Tris-Glycine buffer, PVDF membrane (0.45 μm); Blocking Buffer and antibody diluent: 2.5% skim milk/2.5% BSA in TBST, blocking 1 hour at RT; Primary antibody: 1 μg/mL, overnight at 2-8°C; Secondary antibody: anti-rabbit-HRP (1/10,000) 2.5 hours at RT; Detection: Chemiluminiscence.
The protein named TrkB (also named Neurotrophic tyrosine kinase receptor type 2 (NTRK2), GP145-TrkB or Tropomyosin-related kinase B is a receptor tyrosine kinase involved in the development and the maturation of the central and the peripheral nervous systems and is important in the regulation of neuron survival, proliferation, migration, differentiation, and synapse formation and plasticity. TrkB may also play a role in neutrophin-dependent calcium signaling in glial cells and mediate communication between neurons and glia. TrkB is the primary receptor for BDNF (brain-derived neurotrophic factor. TrkB also binds NT4 and NT3 but less efficiently. Upon ligand-binding, the receptor undergoes homodimerization, autophosphorylation and activation. TrkB activation recruits, phosphorylates and/or activates several downstream effectors including SHC1, FRS2, SH2B1, SH2B2 and PLCG1 that each regulate distinct overlapping signaling cascades within cells. Through SHC1, FRS2, SH2B1, SH2B2, these activate the GRB2-Ras-MAPK cascade that regulates, for instance, neuronal differentiation including neurite outgrowth. These same effectors also control the Ras-PI3 kinase-AKT1 signaling cascade that mainly regulates growth and survival. TrkB, via activation of PLCG1 and the downstream protein kinase C-regulated pathways, also controls synaptic plasticity, and thus plays a role in learning and memory by regulating both short term synaptic function and long-term potentiation. PLCG1 also leads to NF-Kappa-B activation and the transcription of genes involved in cell survival. One such consequence is that PLCG1 activation via TrkB is able to suppress anoikis, the apoptosis resulting from loss of cell-matrix interactions. (Reference: www.uniprot.org)
Affinity-purified
Polyclonal
IgG
WB
Rabbit
120 kDa
Synthetic peptide immunogen, AKASPV[pY]LDILG
Human
Chicken, Human, Mouse, Rat
Spin vial briefly before opening. Reconstitute in 50 µL sterile-filtered, ultrapure water. Centrifuge to remove any insoluble material. Final buffer contains no preservatives. Store lyophilized antibody at 2-8°C. After reconstitution divide into aliquots and store at -20°C for long-term storage. Store at 2-8°C short-term (up to 4 weeks) with an appropriate antibacterial agent. Avoid repetitive freeze/thaw cycles.
Lyophilized
Affinity purified, and absorbed.
PBS, pH 7.2-7.6, 0.1% trehalose, without preservatives.
WB: 0.5-2 µg/mL
High skim milk concentration (5%) causes suppression of pY817 signal, suggesting that 5% skim milk should be avoided as blocking buffer and antibody diluent. Strongest signal is obtained in 5% BSA blocking buffer, however, many non-specific bands are present. An equal mixture of skim milk and BSA (2.5% each) appears to provide the best compromise between signal and noise. However, optimization of blocking condition is recommended for each particular sample, with excess of BSA over skim milk likely to be beneficial for best results.
Unconjugated
Antibody detects a clear band in retinoic acid (RA) and BDNF-treated NSC34 cell lysates at ~120 kDa only. Human TrkB (pY817). Antibody has been shown to be specific for TrkB phosphorylated on tyrosine 817 by phospho-peptide absorption dot blots, and on cell lysates from cell lines induced with retinoic acid and BDNF. Antibody detects a clear band in retinoic acid (RA) and BDNF-treated NSC34 cell lysates at ~120 kDa only, demonstrating that the phosphorylated TrkB receptor is being detected.
While not fully tested, this antibody may detect phosphorylated TrkA (pY791/794, human/rodent) and TrkC (pY834/820/859, human/mouse/rat) due to high degree of amino acid homology surrounding the phosphorylation site.
