Phosphoamino Acid Analysis

Bartholomew.M. Sefton1

1 The Salk Institute, San Diego, Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.5
DOI:  10.1002/0471143030.cb1405s03
Online Posting Date:  May, 2001
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Abstract

Proteins involved in signal transduction are often phosphorylated. Determination of the specific amino acid residue(s) involved is used in characterizing the particular pathway. Partial acid hydrolysis of phosphorylated proteins followed by two‐dimensional thin layer chromatography is used to identify the phosphorylated residues of the protein as phosphoserine, phosphothreonine, or phosphotyrosine. Mild alkaline hydrolysis is used to enhance detection of phosphothreonine and phosphotyrosine.

     
 
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Table of Contents

  • Basic Protocol 1: Acid Hydrolysis and Two‐Dimensional Electrophoretic Analysis of Phosphoamino Acids
  • Alternate Protocol 1: Alkali Treatment to Enhance Detection of TYR‐ and THR‐Phosphorylated Proteins Blotted onto Filters
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Acid Hydrolysis and Two‐Dimensional Electrophoretic Analysis of Phosphoamino Acids

  Materials
  • 32P‐labeled phosphoprotein (unit 14.4)
  • India ink solution: 1 µl/ml India ink in TBS (unit 14.4)/0.02% (v/v) Tween 20, pH 6.5 (prepare fresh or store indefinitely at room temperature); or radioactive or phosphorescent alignment markers
  • 6 M HCl
  • Phosphoamino acid standards mixture (see recipe)
  • pH 1.9 electrophoresis buffer (see recipe)
  • pH 3.5 electrophoresis buffer (see recipe)
  • 0.25% (w/v) ninhydrin in acetone in a freon (aerosol, gas‐driven) atomizer/sprayer
  • PVDF membrane (Immobilon‐P, Millipore)
  • 110° oven
  • Screw‐cap microcentrifuge tubes
  • 20 cm × 20 cm × 100 µm glass‐backed cellulose thin‐layer chromatography plate (EM Sciences)
  • Large blotter: two 25 × 25–cm layers of Whatman 3MM paper sewn together at the edges, with four 2‐cm holes that align with the origins on the TLC plate
  • Glass tray or plastic box
  • Whatman 3MM paper
  • Thin‐layer electrophoresis apparatus (e.g., HTLE 7000, CBS Scientific)
  • Fan
  • Small blotters: 4 × 25–cm, 5 × 25–cm, and 10 × 25–cm pieces of Whatman 3MM paper
  • 50° to 80°C drying oven
  • Sheets of transparency film for overhead projector
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), and autoradiography (unit 6.3)

Alternate Protocol 1: Alkali Treatment to Enhance Detection of TYR‐ and THR‐Phosphorylated Proteins Blotted onto Filters

  • 1 M KOH
  • TN buffer: 10 mM Tris⋅Cl (pH 7.4 at room temperature)/0.15 M NaCl
  • 1 M Tris⋅Cl, pH 7.0 at room temperature
  • Covered plastic container (e.g., Tupperware box)
  • 55°C oven or water bath
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Figures

Videos

Literature Cited

Literature Cited
   Contor, L., Lamy, F. and Lecocq, R.E. 1987. Use of electroblotting to detect and analyze phosphotyrosine containing peptides separated by two‐dimensional gel electrophoresis. Anal. Biochem. 160:414‐420.
   Cooper, J.A. and Hunter, T. 1981. Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins. Mol. Cell. Biol. 1:394‐407.
   Hunter, T. and Sefton, B.M. 1980. The transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Natl. Acad. Sci. U.S.A. 77:1311‐1315.
Key Reference
   Kamps, M.P. and Sefton, B.M. 1989. Acid and base hydrolysis of phosphoproteins bound to Immobilon facilitates the analysis of phosphoamino acids in gel‐fractionated proteins. Anal. Biochem. 176:22‐27.
  Discusses all of the variables involved in subjecting filter‐bound proteins to acid and base hydrolysis.
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