Cell Signaling Technology

Product Pathways - PathScan Multiplex WB Cocktails

PathScan® Bcr/Abl Activity Assay: Phospho-c-Abl, Phospho-Stat5 and Phospho-CrkL Multiplex Western Detection Kit #7130

Kit Includes Applications Reactivity Quantity
PathScan® Bcr/Abl Activity Assay: Phospho-c-Abl, Phospho-Stat5 and Phospho-CrkL Multiplex Western Detection Cocktail #5300 W H 200 µl
Treated and Untreated Cell Extracts 50 µl
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl
Anti-biotin, HRP-linked Antibody #7075 100 µl
20X LumiGLO® Reagent and 20X Peroxide #7003 5 ml
Biotinylated Protein Ladder Detection Pack #7727 100 µl

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

Description

The Pathscan® Multiplex Western Detection Kit offers a unique method to assay the inhibition of multiple proteins on one membrane without stripping and reprobing. This method saves the user valuable time while increasing accuracy and minimizing reagent waste. The system allows the user to simultaneously detect the inhibition of phosphorylation of c-Abl, Stat5 and CrkL proteins in response to STI-571. The kit also includes eIF4E antibody to control protein loading. In addition, each Pathscan® Multiplex Western Detection Kit contains treated and untreated cell lysates and the Phototope®-HRP Western Detection System. The kit includes enough primary and secondary antibodies to perform five Western blot experiments.

Specificity / Sensitivity

Each phospho-antibody in this kit recognizes only the phosphorylated form of its specific target. The eIF4E control antibody detects total levels of eIF4E to determine protein loading. All of the antibodies detect endogenous levels of the target proteins.

Western Blotting

Western Blotting

Western blot analysis of extracts from K562 cells, untreated or STI-571-treated, using PathScan® Bcr/Abl Activity Assay: Multiplex Western Detection Kit to detect the inhibition of phospho-Bcr-Abl, phospho-Stat5 and phospho-CrkL.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with synthetic peptides. Antibodies are purified by protein A and peptide affinity chromatography.

Background

STI-571 (also known as Imatinib mesylate and Gleevec™) is a tyrosine kinase (TK) inhibitor that is a relatively specific ATP-binding site antagonist of Bcr-Abl, PDGF receptor and c-Kit TKs (1-3). Results are encouraging in CML clinical trials, and STI-571 has become a paradigm for targeted cancer therapeutics (4-6). Signal transduction through phospho-tyrosine pathways has been studied extensively, and tyrosine phosphorylation has been linked to multiple cell growth and differentiation pathways (7-9). Because the observed leukemic state of CML is dependent on the intact Bcr-Abl tyrosine kinase activity, extensive work has been done to identify substrates of Bcr-Abl and thus possible mechanisms leading to a myeloid expansion. Many groups have characterized prominent tyrosine-phosphorylated protein substrates in both CML blasts and Bcr-Abl-expressing cell lines, including SHIP, c-cbl, Dok, SHC and CrkL (10-15). In addition, key signal transduction pathways involving PI3 kinase, Ras, Myc and Stat5 are also activated in a Bcr-Abl kinase-dependent manner (16).

  1. Buchdunger, E. et al. (1996) Cancer Res 56, 100-4.
  2. Heinrich, M.C. et al. (2000) Blood 96, 925-32.
  3. Druker, B.J. et al. (1996) Nat Med 2, 561-6.
  4. Mauro, M.J. and Druker, B.J. (2001) Curr Oncol Rep 3, 223-7.
  5. Druker, B.J. et al. (2001) N Engl J Med 344, 1031-7.
  6. Druker, B.J. et al. (2001) N Engl J Med 344, 1038-42.
  7. Blume-Jensen, P. and Hunter, T. (2001) Nature 411, 355-65.
  8. Ullrich, A. and Schlessinger, J. (1990) Cell 61, 203-12.
  9. Cantley, L.C. et al. (1991) Cell 64, 281-302.
  10. ten Hoeve, J. et al. (1994) Blood 84, 1731-6.
  11. Matsuguchi, T. et al. (1994) J Biol Chem 269, 5016-21.
  12. Carpino, N. et al. (1997) Cell 88, 197-204.
  13. Sattler, M. et al. (1997) Oncogene 15, 2379-84.
  14. Di Cristofano, A. et al. (1998) J Biol Chem 273, 4827-30.
  15. Wisniewski, D. et al. (1999) Blood 93, 2707-20.
  16. Kabarowski, J.H. and Witte, O.N. (2000) Stem Cells 18, 399-408.

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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.

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