Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Tyrosine Kinase / Adaptors

ALK (D5F3) XP® Rabbit mAb #3633

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P IF-IC F H Endogenous 220 (ALK), 80 (NPM-ALK), 117 (EML4-ALK v1), 86 (EML4-ALK v3) Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

ALK (D5F3) XP® Rabbit mAb detects endogenous levels of total ALK protein as well as ALK fusion proteins, such as EML4-ALK variants and NPM-ALK, even at low levels. This antibody does not cross-react with other family members.

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein corresponding to residues in the carboxy terminus of human ALK.

Western Blotting

Western Blotting

Western blot analysis of extracts from NCI-H2228 and NCI-H3122 cells using ALK (D5F3) XP® Rabbit mAb. Variants denoting fusions of different EML4 exons (v1 or v3) are indicated.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded anaplastic large cell lymphoma (ALCL) using ALK (D5F3) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma with high (left) and low levels (right) of ALK expression using ALK (D5F3) XP® Rabbit mAb.


Flow Cytometry

Flow Cytometry

Flow cytometric analysis of DU 145 cells (blue) and KARPAS-299 cells (green) using ALK (D5F3) XP® Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of KARPAS-299 cells (left), NCI-H2228 cells (center) and DU 145 cells (right), using ALK (D5F3) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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).

Investigators have identified ALK translocations with other fusion partners, such as TRK-fused gene (TFG) and KIF5B, which have also been associated with NSCLC (6,7). In particular, the EML4-ALK fusion protein has been found in 3-7% of NSCLC samples (6-14).

  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.
  9. Takeuchi, K. et al. (2009) Clin Cancer Res 15, 3143-9.
  10. Palmer, R.H. et al. (2009) Biochem J 420, 345-61.
  11. Horn, L. and Pao, W. (2009) J Clin Oncol 27, 4232-5.
  12. Rodig, S.J. et al. (2009) Clin Cancer Res 15, 5216-23.
  13. Mino-Kenudson, M. et al. (2010) Clin Cancer Res 16, 1561-71.
  14. Kwak, E.L. et al. (2010) N Engl J Med 363, 1693-703.

Application References

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Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

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