Figure 1: Phosphorylation of ALK and NPM-ALK is detected by PathScan® Phospho-ALK (Tyr1586) Sandwich ELISA Kit #7159 (top, right) in Karpas299 cells lysed in the presence of phosphatase inhibitors (phospho-lysate). In contrast, a low level of phospho-ALK is detected in Karpas299 cells lysed in the absence of phosphatase inhibitors (nonphospho-lysate). Similar levels of total ALK from either nonphospho- or phospho-lysates are detected by PathScan® Total ALK Sandwich ELISA Kit #7322 (top, left). Absorbance at 450 nm is shown in the top figure, while the corresponding Western blots, using Phospho-ALK (Tyr1586) (3B4) Rabbit mAb #3348 (right) or a ALK (C26G7) Rabbit mAb #3333 (left) are shown in the bottom figure. Cell Line Source: Dr Abraham Karpas at the University of Cambridge.
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 inhibitors to the lysis buffer. Cell Line Source: Dr Abraham Karpas at the University of Cambridge.
CST's PathScan® Phospho-ALK (Tyr1586) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of ALK and the NPM-ALK fusion protein when phosphorylated at Tyr1586. A phospho-ALK (Tyr1586) Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, phospho-ALK and phospho-NPM-ALK are captured by the coated antibody. Following extensive washing, an ALK Mouse Antibody* is added to detect the captured phospho-ALK and phospho-NPM-ALK fusion protein. 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 ALK and NPM-ALK fusion protein phosphorylated at Tyr1586.
* Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Phospho-ALK (Tyr1586) Sandwich ELISA Kit #7159 detects endogenous levels of ALK and the NPM-ALK fusion protein when phosphorylated at Tyr1586. High levels of phospho-ALK (Tyr1586) and the 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 nonphospho-lysates from Karpas299 cells when lysed without the addition of phosphatase inhibitors* to the lysis buffer. Total ALK (phospho and nonphospho) detected by PathScan® Total ALK Sandwich ELISA Kit #7322 and Western analysis, remain unchanged (Figure 1).
* Phosphatase inhibitors includes sodium pyrophosphate, β-glycerophosphate and Na3VO4.
This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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).
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