Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-ALK (Tyr1604) Sandwich ELISA Kit #7324

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Kit Includes Volume Solution Color
P-ALK (Y1604) Ab Coated Microwells 96 tests
ALK detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

Reactivity Key:
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

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 #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 phospho-ALK (Tyr1604) or phospho-NPM-ALK proteins.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

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.

ELISA - Western correlation

ELISA - Western correlation

Figure 1: Constitutive phosphorylation of ALK or 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-ALK protein is detected in Karpas299 cells lysed without addition of phosphatase inhibitors to the lysis buffer (nonphospho-lysate). However, similar levels of total 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.

Sensitivity

Sensitivity

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).

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).

  1. Stoica, G.E. et al. (2001) J Biol Chem 276, 16772-9.
  2. Iwahara, T. et al. (1997) Oncogene 14, 439-49.
  3. Morris, S.W. et al. (1997) Oncogene 14, 2175-88.
  4. Morris, S.W. et al. (1994) Science 263, 1281-4.
  5. Bai, R.Y. et al. (1998) Mol Cell Biol 18, 6951-61.
  6. Rikova, K. et al. (2007) Cell 131, 1190-203.
  7. Takeuchi, K. et al. (2008) Clin Cancer Res 14, 6618-24.
  8. Soda, M. et al. (2007) Nature 448, 561-6.

Application References

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