Product Pathways - PathScan ELISA

PathScan® Total ALK Sandwich ELISA Kit #7322

PhosphoSitePlus^® protein, site, and accession data: ALK

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Kit Includes	Volume	Solution Color
ALK Ab Coated Microwells	96 tests
ALK Detection Ab	11 ml	Green
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

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

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

Description

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 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 Detection Antibody is added to detect the captured ALK and NPM-ALK proteins. 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 total ALK and NPM-ALK proteins.* Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

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.

ELISA - Western correlation

Figure 1: Constitutive phosphorylation of ALK and 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-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 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.

Sensitivity

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.

Specificity

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.

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). A distinct ALK oncogenic fusion protein involving ALK and EML4 has been described from a non-small cell lung cancer cell line and corresponding fusion transcripts are 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 (6,7).

Application References

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Protocols

7322:: Sandwich ELISA

Companion Products

This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.