Revision 1
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IF-IC, FC-FP

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

80 (NPM-ALK), 220 (ALK)

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q9UM73

Entrez-Gene Id:

238

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunofluorescence (Immunocytochemistry) 1:400
Flow Cytometry (Fixed/Permeabilized) 1:200

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb recognizes endogenous levels of ALK protein only when phosphorylated at Tyr1507 (equivalent to Tyr567 of NPM-ALK).

Species Reactivity:

Human

Species predicted to react based on 100% sequence homology

Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr1507 of human ALK 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 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).
Phosphorylation of ALK on Tyr1507 was identified at Cell Signaling Technology using PhosphoScan®, an LC-MS/MS platform used for phosphorylation site discovery (6). Phosphorylation of ALK at Tyr1507 (Tyr567 in NPM-ALK) has been shown to be important for interaction with the adaptor proteins Shc, FRS2-α, and FRS2-β (9,10).

  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. Turner, S.D. et al. (2007) Cell Signal 19, 740-7.
  10. Chikamori, M. et al. (2007) Oncogene 26, 2950-4.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 1
#14678

Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb

Western Blotting Image 1: Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb Expand Image
Western blot analysis of extracts from KARPAS-299 cells, untreated (-) or treated with Crizotinib #4401 (1 μM, indicated times), using Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb (upper) and ALK (C26G7) Rabbit mAb #3333 (lower). KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
Immunoprecipitation Image 1: Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb Expand Image
Immunoprecipitation of phospho-ALK from KARPAS-299 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb. Western blot analysis was performed using Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb. KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
Immunofluorescence Image 1: Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of KARPAS-299 cells, untreated (left) or treated with Crizotinib #4401 (1 μM, 2hr; center), and HeLa cells (right), using Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
Flow Cytometry Image 1: Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb Expand Image
Flow cytometric analysis of KARPAS-299 cells, untreated (green) or treated with Crizotinib #4401 (1 μM, 2 hr, 37ºC; blue), using Phospho-ALK (Tyr1507) (D6F1V) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody. KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.