Figure 1. Relationship between protein concentration of lysates from untreated and hβ-NGF-treated 3T3/TrkA cells and immediate light generation with chemiluminescent substrate is shown. After starvation, 3T3/TrkA cells (85% confluent) were treated with Human β-Nerve Growth Factor (hβ-NGF) #5221 (100 ng/ml, 2 min) at 37°C, and lysed with 1X cell lysis buffer (10X Cell Lysis Buffer #9803). Graph inset corresponding to the shaded area shows high sensitivity and a linear response at the low protein concentration range.
|Product Includes||Volume||Solution Color|
|TrkA 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||5.5 ml||Green|
|HRP Diluent||5.5 ml||Red|
|Luminol/Enhancer Solution||3 ml|
|Stable Peroxide Buffer||3 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|
The PathScan® Phospho-TrkA (Tyr490) Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of TrkA when phosphorylated at Tyr490 with a chemiluminescent readout. Chemiluminescent ELISAs often have a wider dynamic range and higher sensitivity than conventional chromogenic detection. This chemiluminescent ELISA, which is offered in low volume microplates, shows increased signal and sensitivity while using a smaller sample size. 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 Phospho-TrkA (Tyr490) Rabbit Detection Antibody is added to detect phospho-TrkA protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of TrkA phosphorylated at Tyr490.
Antibodies in kit are custom formulations specific to kit.
NOTE: Refer to product-specific datasheets for assay incubation temperature. This chemiluminescent ELISA is offered in low volume microplates. Only 50 µl of samples or reagents are required in each microwell.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
posted November 2013
Protocol Id: 66
PathScan® Phospho-TrkA (Tyr490) Chemiluminescent Sandwich ELISA Kit #7024 detects endogenous levels of TrkA when phosphorylated at Tyr490 in human 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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