Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total TrkA Sandwich ELISA Kit #7208

Kit Includes Volume Solution Color
TrkA mAb coated microwells 96 tests
TrkA Detection Ab 11 milliliters Green
Anti-rabbit IgG HRP-Linked Ab 11 milliliters Red
TMB Substrate 11 milliliters Colorless
STOP Solution 11 milliliters Colorless
Sealing Tape 2 sheets
20X Wash Buffer 25 milliliters Colorless
Sample Diluent 25 milliliters Blue
Cell Lysis Buffer (10X) # 9803 15 milliliters Yellowish

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

CST's PathScan® Total TrkA Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects transfected levels of total TrkA protein. A TrkA Mouse mAb #4614* has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-TrkA proteins are captured by the coated antibody. Following extensive washing, TrkA Rabbit Antibody #4598* is added to detect both the captured phospho- and nonphospho-TrkA protein. Anti-rabbit IgG, HRP-linked Antibody #7074* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of total TrkA protein.* Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Total TrkA Sandwich ELISA Kit #7208 detects transfected levels of total TrkA Protein. As shown in Figure 1, using PathScan® Phospho-TrkA (Tyr490) ELISA Kit #7210, a significant induction of Phospho-TrkA (Tyr490) is detected in 3T3/TrkA cells treated with NGF. The levels of total TrkA (phospho and nonphospho) detected by PathScan® Total TrkA Sandwich ELISA Kit #7208 remain unchanged. In Figure 3, Western blot analysis of protein captured in the TrkA mouse mAb #4614 coated microwell shows a major band corresponding to the TrkA protein. Using this kit, TrkA protein can also be detected in K562 cells.

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of lysates from untreated and NGF-treated 3T3/TrkA cells and kit assay optical density readings is shown. After starvation, 3T3/TrkA cells (85% confluence) were treated with NGF (100 ng/ml) for 2 min at 37?C, and then lysed.

ELISA - Western correlation

ELISA - Western correlation

Figure 1: Treatment of 3T3/TrkA cells with NGF stimulates phosphorylation of TrkA at Tyr490, detected by PathScan® Phospho-TrkA (Tyr490) Sandwich ELISA kit #7210, but does not affect the level of total TrkA detected by PathScan® Total TrkA Sandwich ELISA kit #7208. OD 450 readings are shown in the top figure, while the corresponding Western blots using Phospho-TrkA (Tyr490) Antibody #9141 (right panel) or TrkA Rabbit mAb #2505 (left panel), are shown in the bottom figure. The human TrkA is expressed in 3T3/TrkA cells.

Specificity

Specificity

Figure 3: Kit specificity demonstrated by Western blot analysis of the ELISA-well captured protein. Lysates were prepared from NIH/3T3 cells transfected with human TrkA and incubated in wells coated with capture antibody #4614. Wells were then washed, and captured protein was solubilized in SDS gel loading buffer. 3T3/TrkA lysate (lane 1) and captured protein (lane 2) were analyzed by Western blot using TrkA antibody #2505. A band corresponding to the TrkA protein is detected in the captured material (lane 2).


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).

  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.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

Companion Products

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

Products