Phosphoamino Acid Analysis

Bartholomew M. Sefton1

1 The Salk Institute, San Diego
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 13.3
DOI:  10.1002/0471140864.ps1303s04
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

It is often valuable to identify the phosphorylated residue in a protein. This unit presents a protocol for partial acid hydrolysis of proteins phosphorylated at serine, threonine, or tyrosine, followed by two‐dimensional thin‐layer electrophoresis of the labeled phosphoamino acid. Phosphothreonine and phosphotyrosine are more stable to hydrolysis in alkali than are RNA and phosphoserine. Therefore, an alternate procedure using mild alkaline hydrolysis of protein samples to enhance the detection of phosphothreonine and phosphotyrosine is also provided.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

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

  Materials
  • 32P‐labeled phosphoprotein (unit 13.2)
  • India ink solution: 1 µl/ml India ink in recipeTBS (see recipe)/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 ( appendix 2E)
  • recipePhosphoamino acid standards mixture (see recipe)
  • recipepH 1.9 electrophoresis buffer (see recipe)
  • recipepH 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°C 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 10.1), electroblotting onto PVDF membranes (unit 10.7), and staining membranes (unit 10.8)

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 ( appendix 2E)
  • Covered plastic container (e.g., Tupperware box)
  • 55°C oven or water bath
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library