Figure 1. Treatment of 3T3/TrkB cells with BDNF stimulates tyrosine-phosphorylation of TrkB, detected by PathScan® Phospho-TrkB (Tyr516) Sandwich ELISA Kit #7111, but does not affect the levels of total TrkB detected by PathScan® Total TrkB Sandwich ELISA Kit #7106. Absorbance at 450 nm is shown in the top figure, while the corresponding Western blots using Phospho-TrkA (Tyr490)/TrkB (Tyr516) (C35G9) Rabbit mAb #4619 (right panel) or TrkB (80E3) Rabbit mAb #4603 (left panel), are shown in the bottom figure. Human TrkB is transfected and expressed in NIH/3T3 cells.
Figure 2. The relationship between protein concentration of lysates from untreated and BDNF-treated 3T3/TrkB cells and the absorbance at 450 nm is shown. 3T3/TrkB cells (85% confluence) were starved and treated with BDNF (100 ng/ml) for 2 min at 37ºC, and then lysed.
|Product Includes||Volume||Solution Color|
|TrkB Mouse mAb Coated Microwells||96 tests|
|Phospho-TrkA (Tyr490)/TrkB (Tyr516) Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-TrkB (Tyr516) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects transfected levels of TrkB when phosphorylated at Tyr516. A TrkB mouse antibody has been coated onto the microwells. After incubation with cell lysates, TrkB (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-TrkB (Tyr516) rabbit detection antibody is added to detect tyrosine phosphorylation of the captured TrkB protein. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of TrkB phosphorylated on Tyr516.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-TrkB (Tyr516) Sandwich ELISA Kit #7111 detects transfected levels of TrkB protein when Tyr516 is phosphorylated (see Figure 1). The kit sensitivity is shown in figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
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).
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PathScan is a trademark of Cell Signaling Technology, Inc.