Detecting Tyrosine‐Phosphorylated Proteins by Western Blot Analysis

Sansana Sawasdikosol1

1 Mount Sinai School of Medicine, New York, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 11.3
DOI:  10.1002/0471142735.im1103s89
Online Posting Date:  April, 2010
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The development of monoclonal antibodies (mAbs) that recognize nearly all of the phosphorylated tyrosine residues, irrespective of the surrounding sequences, enables researchers to detect the phosphorylation state of proteins through the use of anti‐phosphotyrosine western blotting. The availability of this simple, reliable, nonradioactive and yet sensitive method created a boom in signal transduction research. While the methodology of how to perform an anti‐phosphotyrosine western blot remains unchanged since the procedure became widely used in the early part of 1990s, steady improvements in reagents and detection technologies have allowed researchers to detect tyrosine phosphorylation quantitatively, at unprecedented sensitivity. In addition to the improvements in the western blot–based systems, powerful new phosphotyrosine detection platforms, based on proteomic technologies, are emerging rapidly. This unit will describe in detail the steps needed to perform the standard anti‐phosphotyrosine western blot analysis. Curr. Protoc. Immunol. 89:11.3.1‐11.3.11. © 2010 by John Wiley & Sons, Inc.

Keywords: tyrosine‐phosphorylated proteins; tyrosine kinase substrates; anti‐phosphotyrosine western blot

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Jurkat T cells (American Type Culture Collection [ATTC])
  • RPMI‐1640 medium (Mediatech) and fetal bovine serum (Life Technologies)
  • RPMI 1640 medium (Mediatech)
  • Anti‐CD3ɛ, clone OKT3.14 (eBioscience)
  • Rabbit anti‐mouse Ig, unlabeled (Southern Biotech)
  • 1× lysis buffer, ice‐cold (see recipe)
  • 2× SDS sample buffer (see recipe in unit 8.4)
  • Immobilon‐P Polyvinylidene Fluoride (PVDF) membrane, 0.45‐µm pore size (Millipore)
  • 4% BSA PVDF blocking buffer (see recipe)
  • Tris‐buffered saline containing Tween 20 (TBST; see recipe)
  • 0.02% sodium azide (NaN 3)
  • Unlabeled and HRP‐conjugated anti‐phosphotyrosine antibody, clone 4G10 (Millipore Corporation, Billerica, MA)
  • HRP‐conjugated, sheep anti‐mouse Ig (GE Healthcare)
  • Enhanced chemiluminescence (ECL) reagents, Western Lightning (Perkin Elmer Life Sciences)
  • PVDF membrane stripping solution (see recipe)
  • Infrared dye‐conjugated secondary antibody (Li‐Cor Biosciences), optional
  • 75‐ or 175‐cm2 flasks
  • Centrifuge
  • Vacuum apparatus
  • 15‐ml conical centrifuge tubes
  • 1.5‐ml microcentrifuge tubes
  • Vortex
  • 37°C water bath
  • Microcentrifuge
  • Rocker platform or orbital rotating platform, 4°C and room temperature
  • Small plastic container with a flat bottom large enough to allow the membrane to lie flat without touching the side walls of the container
  • Forceps
  • Paper towels
  • Saran Wrap plastic sheet
  • Autoradiographic cassette
  • Scientific imaging film (GE Healthcare's Hyperfilm ECL or Kodak's X‐OMAT)
  • Temperature‐controlled incubator capable of maintaining 50°C, (Boekel Scientific)
  • CCD camera‐equipped imaging device to detect chemiluminescence signals (such as GE Healthcare's ImageQuant RT ECL or Alpha Innotech's FluorChem HD2), optional
  • Odyssey Near Infrared imager (Li‐Cor Biosciences), optional
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and the transfer of electrophoretically resolved proteins onto PVDF membrane (unit 8.10)
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Literature Cited

Literature Cited
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   Brunswick, M., Samelson, L.E., and Mond, J.J. 1991. Surface immunoglobulin crosslinking activates a tyrosine kinase pathway in B cells that is independent of protein kinase. Proc. Natl. Acad. Sci. U.S.A. 88:1311‐1314.
   Ding, S.J., Qian, W.J., and Smith, R.D. 2007. Quantitative proteomic approaches for studying phosphotyrosine signaling. Exp. Rev. Proteomics 4:13‐23.
   Gold, M.R., Law, D.A., and DeFranco, A.L. 1990. Stimulation of protein tyrosine phosphorylation by the B‐lymphocyte antigen receptor. Nature 345:810‐813.
   Horak, I.D., Gress, R.E., Lucas, P.J., Horak, E.M., Waldmann, T.A., and Bolen, J.B. 1991. T‐lymphocyte interleukin 2‐dependent tyrosine protein kinase signal transduction involves the activation of p56lck. Proc. Natl. Acad. Sci. U.S.A. 88:1996‐2000.
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   Mahlknecht, U., Ottmann, O.G., and Hoelzer, D. 2001. Far‐Western based protein‐protein interaction screening of high‐density protein filter arrays. J. Biotechnol. 88:89‐94.
   Mandell, J.W. 2003. Phosphorylation state‐specific antibodies: Applications in investigative and diagnostic pathology. Am. J. Pathol. 163:1687‐1698.
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   Zhang, Y., Wolf‐Yadlin, A., and White, F.M. 2007. Quantitative proteomic analysis of phosphotyrosine‐mediated cellular signaling networks. Methods Mol. Biol. 359:203‐212.
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