| 货号 | 7322C |
| 描述 | CSTs PathScan® Total ALK Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total ALK and NPM-ALK fusion protein. An ALK Capture antibody has been coated onto the microwells. After incubation with cell lysates, ALK and NPM-ALK proteins (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, an ALK Detection Antibody is added to detect the captured ALK and NPM-ALK proteins. Anti-mouse IgG, HRP-linked Antibody #7076* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of total ALK and NPM-ALK proteins. * Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Total ALK Sandwich ELISA Kit #7322 detects endogenous levels of total ALK and NPM-ALK fusion proteins. High levels of phospho-ALK (Tyr1604) protein and phospho-NPM-ALK fusion protein are detected in Karpas299 cells where ALK and NPM-ALK are constitutively phosphorylated (Figure 1). These high levels are abolished in Karpas299 cells lysed without addition of phosphatase inhibitors* to the lysis buffer. The levels of total ALK protein (phospho and nonphospho) detected by PathScan® Total ALK Sandwich ELISA Kit #7322 remain unchanged (Figure 1). Western analysis of protein captured in microwells coated with ALK antibody shows two bands corresponding to both ALK protein and NPM-ALK fusion protein (Figure 3). 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 | Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor for pleiotrophin (PTN), a growth factor involved in embryonic brain development (1-3). In ALK-expressing cells, PTN induces phosphorylation of both ALK and the downstream effectors IRS-1, Shc, PLCγ, and PI3 kinase (1). ALK was originally discovered as an NPM (nucleophosmin)-ALK fusion protein produced by a translocation (4). The NPM-ALK fusion protein is a constitutively active, oncogenic tyrosine kinase associated with anaplastic lymphoma (4). Activation of PLCγ by NPM-ALK has been suggested to be a crucial step for its mitogenic activity and may be important in the pathogenesis of anaplastic lymphomas (5). A distinct ALK oncogenic fusion protein involving ALK and EML4 has been described from a non-small cell lung cancer cell line, with corresponding fusion transcripts present in some cases of lung adenocarcinoma. The short, amino-terminal region of the microtubule-associated protein EML4 is fused to the kinase domain of ALK (6,7). |
| 存放说明 | 4C |
| 参考文献 | Stoica, G.E. et al. (2001) J Biol Chem 276, 16772-9. Iwahara, T. et al. (1997) Oncogene 14, 439-49. Morris, S.W. et al. (1997) Oncogene 14, 2175-88. Morris, S.W. et al. (1994) Science 263, 1281-4. Bai, R.Y. et al. (1998) Mol Cell Biol 18, 6951-61. Rikova, K. et al. (2007) Cell 131, 1190-203. Takeuchi, K. et al. (2008) Clin Cancer Res 14, 6618-24. Soda, M. et al. (2007) Nature 448, 561-6. |
Figure 3. Kit specificity as demonstrated by western analysis of the ELISA microwell captured protein. Human Karpas299 cell lysates were incubated in microwells coated with ALK capture antibody. Following washing, captured protein was solubilized in SDS gel loading buffer. Karpas299 cell lysates (lane 1) and captured protein (lane 2) were analyzed by western blot using the ALK detection antibody. A pair of distinct bands in the captured material (lane 2) correspond to both ALK protein and NPM-ALK fusion protein.图3：试剂盒特异性检测，如图用免疫印迹法对ELISA微孔中捕获的蛋白进行检测。在包被有ALK蛋白捕获抗体的微孔中对人的Karpas299细胞裂解液进行孵育。洗板后，用SDS胶的上样溶液对捕获的蛋白进行溶解。用抗ALK的检测抗体对Karpas299细胞裂解液（第一道）和被捕获蛋白（第二道）进行免疫印迹检测。被捕获的物在对应ALK 蛋白和NPM-ALK 融合蛋白的位置上能检测到两条清晰的带(Figure 3) | |
Figure 2: The relationship between protein concentration of phospho- or nonphospho-lysates and the absorbance at 450 nm is shown. Karpas299 cells were harvested at 106 cells/ml, and lysed with or without addition of phosphatase inhibitor to the lysis buffer. 图2：敏感性检测。在波长450nm处对加入磷酸酶抑制剂处理（含磷酸化蛋白）和未经磷酸酶抑制剂处理的细胞裂解液（不含磷酸化蛋白）进行目的蛋白浓度检测的结果。Karpas299细胞在浓度为106 cells/ml时收集，并加入或不加磷酸酶抑制剂进行裂解处理。 |
