Product Pathways - PathScan ELISA
PathScan® Phospho-ALK (Tyr1586) Sandwich ELISA Kit #7159
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Phospho-ALK (Tyr1586) Rabbit Antibody Coated Microwells | 96 tests | |
| ALK Mouse 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 |
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 (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.
Specificity / Sensitivity
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.
Sandwich ELISA
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.
Sandwich ELISA
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.
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
- 3343 Phospho-ALK (Tyr1586) Antibody
- 3348 Phospho-ALK (Tyr1586) (3B4) Rabbit mAb
- 3341 Phospho-ALK (Tyr1604) Antibody
- 3333 ALK (C26G7) Rabbit mAb
- 7324 PathScan® Phospho-ALK (Tyr1604) Sandwich ELISA Kit
- 7322 PathScan® Total ALK Sandwich ELISA Kit
- 9803 Cell Lysis Buffer (10X)
- 7076 Anti-mouse IgG, HRP-linked Antibody
