Western blot analysis of extracts from NIH/3T3, NIH/3T3-TrkA, NIH/3T3-TrkB and NIH/3T3-TrkC cells, using TrkB Antibody (upper), TrkA Antibody #2505 (middle) and PLCγ1 Antibody #2822 (lower).Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
TrkB Antibody detects transfected levels of total TrkB protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to human TrkB. Antibodies are purified by protein A and peptide affinity chromatography.
The family of Trk receptor tyrosine kinases consists of TrkA, TrkB, and TrkC. While the sequence of these family members is highly conserved, they are activated by different neurotrophins: TrkA by NGF, TrkB by BDNF or NT4, and TrkC by NT3 (1). Neurotrophin signaling through these receptors regulates a number of physiological processes, such as cell survival, proliferation, neural development, and axon and dendrite growth and patterning (1). In the adult nervous system, the Trk receptors regulate synaptic strength and plasticity. TrkA regulates proliferation and is important for development and maturation of the nervous system (2). Phosphorylation at Tyr490 is required for Shc association and activation of the Ras-MAP kinase cascade (3,4). Residues Tyr674/675 lie within the catalytic domain, and phosphorylation at these sites reflects TrkA kinase activity (3-6). Point mutations, deletions, and chromosomal rearrangements (chimeras) cause ligand-independent receptor dimerization and activation of TrkA (7-10). TrkA is activated in many malignancies including breast, ovarian, prostate, and thyroid carcinomas (8-13). Research studies suggest that expression of TrkA in neuroblastomas may be a good prognostic marker as TrkA signals growth arrest and differentiation of cells originating from the neural crest (10).
The phosphorylation sites are conserved between TrkA and TrkB: Tyr490 of TrkA corresponds to Tyr512 in TrkB, and Tyr674/675 of TrkA to Tyr706/707 in TrkB of the human sequence (14). TrkB is overexpressed in tumors, such as neuroblastoma, prostate adenocarcinoma, and pancreatic ductal adenocarcinoma (15). Research studies have shown that in neuroblastomas, overexpression of TrkB correlates with an unfavorable disease outcome when autocrine loops signaling tumor survival are potentiated by additional overexpression of brain-derived neurotrophic factor (BDNF) (16-18). An alternatively spliced truncated TrkB isoform lacking the kinase domain is overexpressed in Wilms’ tumors and this isoform may act as a dominant-negative regulator of TrkB signaling (17).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Explore pathways related to this product.