Equipamentos para Laboratórios | Equipamento para Laboratório

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity:  H

Reactivity Key:  H=Human

Description

The PathScan® Phospho-TrkB (Tyr706/707) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects transfected levels of TrkB when phosphorylated at Tyr706/707. 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 (Tyr706/707) 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 Tyr706/707.

Specificity / Sensitivity

CST's PathScan® Phospho-TrkB (Tyr706/707) Sandwich ELISA Kit detects transfected levels of phospho-TrkB protein when Tyr706/707 is phosphorylated (see Figure 1). The kit sensitivity is shown in figure 2.

Sandwich ELISA

Figure 1: Treatment of 3T3/TrkB cells with BDNF (#3897) stimulates tyrosine-phosphorylation of TrkB, detected by PathScan® Phospho-TrkB (Tyr706/707) Sandwich ELISA Kit #7118, but does not affect the level 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 (Tyr674/675)/TrkB (Tyr706/707) (C50F3) Rabbit mAb #4621 (right panel) or TrkB (80E3) Rabbit mAb #4603 (left panel), are shown in the bottom figure. The human TrkB is transfected and expressed in NIH/3T3 cells.

Sandwich ELISA

Figure 2: The relationship between protein concentration of lysates from untreated and BDNF-treated 3T3/TrkB cells andthe absorbance at 450 nm is shown. After starvation, 3T3/TrkB cells (85% confluence) were treated with BDNF (100 ng/ml) for 2 min at 37°C, and then lysed.

Background

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. TrkA regulates proliferation and is important for development and maturation of the nervous system (1). Phosphorylation at Tyr490 is required for Shc association and activation of the Ras-MAP kinase cascade. Residues Tyr674/675 lie within the catalytic domain, and phosphorylation at this site reflects TrkA kinase activity (2-6). Point mutations, deletions and chromosomal rearrangements (chimeras) cause ligand-independent receptor dimerization and activation of TrkA. Many malignancies including breast, colon, prostate and thyroid carcinomas and acute myeloid leukemia have activated TrkA. Expression of TrkA in neuroblastomas is a good prognostic marker because it signals growth arrest and differentiation of cells originating from the neural crest (1).

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 (7). TrkB is overexpressed in tumors such as neuroblastoma, prostate adenocarcinoma and pancreatic ductal adenocarcinoma. In neuroblastomas overexpression of TrkB correlates with unfavorable disease outcome when autocrine loops signaling tumor survival are potentiated by additional overexpression of brain-derived neurotrophic factor (BDNF). An alternatively spliced truncated TrkB isoform lacking the kinase domain is overexpressed in Wilms’s tumors and this isoform may act as a dominant-negative to TrkB signaling (8).

   1. Pierotti, M.A. and Greco, A. (2006) Cancer Lett 232, 90-8.
   2. Segal, R.A. and Greenberg, M.E. (1996) Annu Rev Neurosci 19, 463-89.
   3. Stephens, R.M. et al. (1994) Neuron 12, 691-705.
   4. Obermeier, A. et al. (1993) EMBO J 12, 933-41.
   5. Obermeier, A. et al. (1994) EMBO J 13, 1585-90.
   6. Yao, R. and Cooper, G.M. (1995) Science 267, 2003-6.
   7. Huang, E.J. and Reichardt, L.F. (2003) Annu. Rev. Biochem. 72, 609-642.
   8. Desmet, C.J. and Peeper, D.S. (2006) Cell. Mol. Life Sci. 63, 755-759.

Protocols

7118:  Sandwich ELISA

Companion Products

    * 3897 Human Brain-Derived Neurotrophic Factor (BDNF)
    * 7106 PathScan® Total TrkB Sandwich ELISA Kit
    * 7108 PathScan® Phospho-TrkB (panTyr) Sandwich ELISA Kit
    * 7111 PathScan® Phospho-TrkB (Tyr516) Sandwich ELISA Kit
    * 4619 Phospho-TrkA (Tyr490)/TrkB (Tyr516) (C35G9) Rabbit mAb
    * 4621 Phospho-TrkA (Tyr674/675)/TrkB (Tyr706/707) (C50F3) Rabbit mAb
    * 4603 TrkB (80E3) Rabbit mAb
    * 4606 TrkB Antibody
    * 4607 TrkB (80G2) Rabbit mAb
    * 4609 Trk (pan) (C17F1) Rabbit mAb
    * 9803 Cell Lysis Buffer (10X)
    * 9808 Phosphate Buffered Saline (PBS-20X)
    * 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
    * 9998 BSA
    * 7004 TMB Substrate
    * 7002 STOP Solution
    * 7074 Anti-rabbit IgG, HRP-linked Antibody