Detection of Phosphorylation by Immunological Techniques

Bartholomew M. Sefton1

1 The Salk Institute, San Diego
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 13.4
DOI:  10.1002/0471140864.ps1304s00
Online Posting Date:  May, 2001
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Abstract

Phosphorylation of unlabeled proteins can be detected using immunological or enzymatic techniques. Anti‐phosphotyrosine antibodies are used with immunoblots to detect tyrosine phosphorylation. This unit presents a protocol employing anti‐phosphotyrosine antibodies with detection by either 125I‐labeled protein A or enhanced chemiluminescence (ECL).

     
 
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Table of Contents

  • Basic Protocol 1: Immunoblotting with Anti‐Phosphotyrosine Antibodies and Detection Using [125I]Protein A
  • Alternate Protocol 1: Detection of Bound Antibodies by Enhanced Chemiluminescence (ECL)
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Immunoblotting with Anti‐Phosphotyrosine Antibodies and Detection Using [125I]Protein A

  Materials
  • Protein sample: Cultured cells, tissue, lysate, or immunoprecipitate
  • Transfer buffer containing 100 µM sodium vanadate
  • recipeBlocking buffer (see recipe)
  • Anti‐phosphotyrosine antibody: 2 µg/ml rabbit polyclonal anti‐phosphotyrosine (UBI) or mouse monoclonal anti‐phosphotyrosine—e.g., py20 (Leinco, ICN Biomedicals, Zymed, Transduction Laboratories) or 4G10 (UBI)—in recipeblocking buffer
  • TN buffer: 10 mM Tris⋅Cl (pH 7.4 at room temperature)/ 0.15 M NaCl ( appendix 2E)
  • TNA solution: TN buffer (recipe above)/0.01% (v/v) sodium azide (store at room temperature)
  • NP‐40 wash solution: 0.05% (v/v) Nonidet P‐40 (NP‐40)/TNA solution (store at room temperature)
  • 0.5 µCi/ml 125I‐labeled protein A (30 mCi/mg, ICN) in recipeblocking buffer
  • India ink solution: 1 µl India ink in TBS (unit 13.3)/0.02% (w/v) Tween 20, pH 6.5 (prepare fresh or store indefinitely at room temperature; optional)
  • Plastic container with lid (e.g., Tupperware box)
  • Blotting paper
  • Additional reagents and solutions for SDS‐PAGE (unit 10.1) and fluorography (unit 10.2)
NOTE: Wear gloves and use blunt‐end forceps to handle membrane.

Alternate Protocol 1: Detection of Bound Antibodies by Enhanced Chemiluminescence (ECL)

  • recipeBlocking buffer (see recipe) without azide
  • 0.05% (v/v) Nonidet P‐40 (NP‐40)/TN buffer (see protocol 1)
  • Horseradish peroxidase–conjugated secondary antibody: anti‐rabbit or anti‐mouse antibody diluted 1/1000 to 1/2000 in recipeblocking bufferwithout azide
  • ECL detection reagents A and B (Amersham)
  • Luminol reagent for ECL detection (Amersham)
  • Oxidizing reagent for ECL detection (Amersham)
  • Nitrocellulose membrane
  • Plastic container slightly larger than the membrane
  • Plastic sheet protector
NOTE: The reagents for ECL may be purchased as a kit, the Enhanced Chemiluminescence Western Blotting Detection System, from Amersham.NOTE: Wear gloves and use blunt‐end forceps to handle membrane.
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Figures

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Literature Cited

Literature Cited
   Frackelton, A.R., Ross, A.H., and Eisen, H.N. 1983. Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus‐transformed and growth factor‐stimulated cells. Mol. Cell. Biol. 3:1343‐1352.
Key References
   Kamps, M.P. and Sefton, B.M. 1988. Identification of multiple novel polypeptide substrates of the v‐src, v‐yes, v‐fps, v‐ros, and v‐erb‐B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene 2:305‐315.
  Examines many of the variables that affect immunoblotting with antibodies to phosphotyrosine.
   Morla, A. and Wang, J.Y.J. 1986. Protein tyrosine phosphorylation in the cell cycle of BALB/c 3T3 fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 83:8191‐8195.
  Establishes the ability of immunoblotting with antibodies to phosphotyrosine to detect the low levels of tyrosine‐phosphorylated proteins in normal cells.
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