Figure 1: Constitutive phosphorylation of NPM-ALK in Karpas299 cells lysed in the presence of phosphatase inhibitors (phospho-lysate) is detected by PathScan® Phospho-ALK (Tyr1604) Sandwich ELISA Kit #7324 (top,right). In contrast, only a low level of phospho-NPM-ALK protein is detected in Karpas299 cells lysed without addition of phosphatase inhibitors to the lysis buffer (nonphospho-lysate). However, similar levels of NPM-ALK protein 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 (Tyr1604) Antibody #3341 (right) or a Total ALK Rabbit mAb #3337 (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 inhibitor to the lysis buffer. Cell Line Source: Dr Abraham Karpas at the University of Cambridge.
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. Cell Line Source: Dr Abraham Karpas at the University of Cambridge.
|7322C||1 Kit (96 assays)|
|Product Includes||Volume||Solution Color|
|ALK Rabbit mAb Coated Microwells||96 tests|
|ALK Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 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|
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.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
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:
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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