Product Pathways - PathScan ELISA
PathScan® Phospho-ALK (Tyr1604) Sandwich ELISA Kit #7324
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Phospho-ALK (Tyr1604) Antibody Coated Microwells | 96 tests | |
| ALK detection Ab | 11 milliliters | Green |
| Anti-mouse IgG HRP-Linked Ab | 11 milliliters | Red |
| TMB Substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| Sealing Tape | 2 sheets | |
| 20X Wash Buffer | 25 milliliters | Colorless |
| Sample Diluent | 25 milliliters | Blue |
| Cell Lysis Buffer (10X) # 9803 | 15 milliliters | Yellowish |
| Phospho-ALK (Tyr1604) Antibody # 3341 | ||
| ALK mouse mAb |
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 (10X), which is stored at –20°C (packaged separately).
Species Cross-Reactivity
H
Reactivity Key: H=Human
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.* Phosphatase inhibitors includes sodium pyrophosphate, β-glycerophosphate and Na3VO4.
Sandwich ELISA
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 #3337 (left panel), are shown in the bottom figure.
Sandwich ELISA
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 an NPM (nucleophosmin)-ALK fusion protein produced by a translocation (4). The NPM-ALK fusion protein is a constitutively active oncogenic tyrosine kinase associated with anaplastic lymphoma (4). Activation of PLCγ by NPM-ALK has been suggested to be a crucial step for its mitogenic activity and may be important in the pathogenesis of anaplastic lymphomas. (5).
- Stoica, G.E. et al. (2001) J. Biol. Chem. 276, 16772-16779.
- Iwahara, T. et al. (1997) Oncogene 14, 439-449.
- Morris, S.W. et al. (1997) Oncogene 14, 2175-2188.
- Morris, S.W. et al. (1994) Science 263, 1281-1284.
- Bai, R.Y. et al. (1998) Mol. Cell Biol. 18, 6951-6961.
Application References
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Companion Products
- 3341 Phospho-ALK (Tyr1604) Antibody
- 3348 Phospho-ALK (Tyr1586) (3B4) Rabbit mAb
- 3343 Phospho-ALK (Tyr1586) Antibody
- 9803 Cell Lysis Buffer (10X)
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 7322 PathScan® Total ALK Sandwich ELISA Kit
- 7159 PathScan® Phospho-ALK (Tyr1586) Sandwich ELISA Kit
- 3333 ALK (C26G7) Rabbit mAb
