PathScan® Total ALK Sandwich ELISA Kit #7322
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Supporting Data
| REACTIVITY | H |
Species Cross-Reactivity Key:
- H-Human
Product Information
Product Description
CST's 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 rabbit 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 mouse detection antibody is added to detect the captured ALK and NPM-ALK proteins. Anti-mouse IgG, HRP-linked Antibody 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.
Protocol
Specificity / Sensitivity
CST's 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.
* Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate and Na3VO4.
Species Reactivity:
Human
Background
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 a nucleophosmin (NPM)-ALK fusion protein produced by a translocation (4). Investigators have found that the NPM-ALK fusion protein is a constitutively active, oncogenic tyrosine kinase associated with anaplastic lymphoma (4). Research literature suggests that activation of PLCγ by NPM-ALK may be a crucial step for its mitogenic activity and involved in the pathogenesis of anaplastic lymphomas (5).A distinct ALK oncogenic fusion protein involving ALK and echinoderm microtubule-associated protein like 4 (EML4) has been described in the research literature from a non-small cell lung cancer (NSCLC) 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 (6-8).
- 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.
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