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.
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.
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).
|Product Includes||Volume||Solution Color|
|TrkA Mouse mAb Coated Microwells||96 tests|
|TrkA Rabbit Detection Antibody||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||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
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 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, a TrkA Rabbit Antibody is added to detect both the captured phospho- and nonphospho-TrkA 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 optical density for this developed color is proportional to the quantity of total TrkA protein.
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® 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 coated microwell shows a major band corresponding to the TrkA protein. Using this kit, TrkA protein can also be detected in K562 cells. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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).
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