| 货号 | 7208C |
| 描述 | CSTs 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. |
| 反应种属 | Human |
| 应用 | ELISA |
| 目标/特异性 | CSTs 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. |
| 供应商 | CST |
| 背景 | 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). |
| 存放说明 | 4C |
| 参考文献 | Huang, E.J. and Reichardt, L.F. (2003) Annu Rev Biochem 72, 609-42. Segal, R.A. and Greenberg, M.E. (1996) Annu Rev Neurosci 19, 463-89. Stephens, R.M. et al. (1994) Neuron 12, 691-705. Marsh, H.N. et al. (2003) J Cell Biol 163, 999-1010. Obermeier, A. et al. (1993) EMBO J 12, 933-41. Obermeier, A. et al. (1994) EMBO J 13, 1585-90. Arevalo, J.C. et al. (2001) Oncogene 20, 1229-34. Reuther, G.W. et al. (2000) Mol Cell Biol 20, 8655-66. Greco, A. et al. (1997) Genes Chromosomes Cancer 19, 112-23. Pierotti, M.A. and Greco, A. (2006) Cancer Lett 232, 90-8. Lagadec, C. et al. (2009) Oncogene 28, 1960-70. Greco, A. et al. (2010) Mol Cell Endocrinol 321, 44-9. Ødegaard, E. et al. (2007) Hum Pathol 38, 140-6. |
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). 图3：试剂盒特异性由对ELISA孔内捕获的蛋白进行Western blot来确定。转染人TrkA的NIH/3T3细胞提取物在包被了捕获抗体#4614的孔板内预孵。然后洗脱孔板，捕获抗体即溶于SDS胶的loading buffer（上样缓冲液）中。3T3/TrkA提取物(列1)和捕获蛋白(列2)使用TrkA Antibody 兔多抗#2505进行Western blot分析。捕获物质中对应于TrkA蛋白的条带在列2中发现。 | |
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. 图1：NGF处理后的3T3/TrkA细胞产生了TrkA的Tyr490磷酸化，为PathScan Phospho-TrkA (Tyr490) Sandwich ELISA kit #7210所检测到，但是却并不产生PathScan Total TrkA Sandwich ELISA kit #7208可检测到的总TrkA水平的变化。OD 450 读数在上图显示，对应的使用Phospho-TrkA (Tyr490) Antibody 兔多抗#9141（右）或TrkA Rabbit mAb 兔单抗#2505（左）的结果在下图显示。人TrkA在3T3/TrkA细胞中表达。 | |
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. 图2：显示未处理和经NGF处理的3T3/TrkA细胞提取液中的蛋白浓度与试剂盒光学浓度读数的关系。饥饿处理后，3T3/TrkA细胞(85% confluence)用100ng/ml 的NGF在37度处理两分钟，然后裂解。 |
