Figure 1: Constitutive phosphorylation of NPM-ALK in Karpas299 cells lysed in the presence of phosphatase inhibitors (phospho-lysate) can be detected by PathScan® Phospho-ALK (Tyr1604) Sandwich ELISA Kit #7324. 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 total NPM-ALK protein from either nonphospho or phospho-lysates can be detected by PathScan® Total ALK Sandwich ELISA Kit #7322. Absorbance at 450 nm is shown in the top figure, while the corresponding Western blots using Phospho-ALK (Tyr1604) Antibody #3341 (right panel) or a total ALK Rabbit mAb #3333 (left panel), are shown in the bottom figure.
Figure 2: The relationship between protein concentration of phospho or non-phospho lysates and the absorbance at 450 nm is shown. Unstarved Karpas299 cells were cultured (106 cells/ml) and lysed with or without addition of phosphatase inhibitor to the lysis buffer (phospho or non-phospho lysate).
|7324C||1 Kit (96 assays)||N/A|
|Product Includes||Volume||Solution Color|
|Phospho-ALK (Tyr1604) Rabbit Ab 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® Phospho-ALK (Tyr1604) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-ALK (Tyr1604) or phospho-NPM-ALK fusion protein. A Phospho-ALK (Tyr1604) Antibody has been coated onto the microwells. After incubation with cell lysates, only phospho-ALK or phospho-NPM-ALK proteins are captured by the coated antibody. Following extensive washing, an ALK Mouse mAb is added to detect the captured phospho-ALK or phospho-NPM-ALK fusion protein. 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 phospho-ALK (Tyr1604) or phospho-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® Phospho-ALK (Tyr1604) Sandwich ELISA Kit #7324 detects endogenous levels of phospho-ALK (Tyr1604) protein or phospho-NPM-ALK fusion protein. As shown in Figure 1, a high level of phosphorylated ALK (Tyr1604) protein or phospho-NPM-ALK fusion protein is detected in Karpas299 cells where ALK or NPM-ALK is constitutively phosphorylated. 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. 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 includes sodium pyrophosphate, β-glycerophosphate and Na3VO4.
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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PathScan is a trademark of Cell Signaling Technology, Inc.