Phosphoamino Acid Analysis

Bartholomew M. Sefton1

1 The Salk Institute, San Diego, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.3
DOI:  10.1002/0471142727.mb1803s40
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. In the case of proteins phosphorylated at serine, threonine, or tyrosine, this is readily accomplished by partial acid hydrolysis in HCl followed by two‐dimensional thin‐layer electrophoresis of the labeled phosphoamino acid, as described here. Phosphothreonine andphosphotyrosine are more stable to hydrolysis in alkali than are RNA andpho sphoserine. Therefore, a protocol for mild alkaline hydrolysis of protein samples is also provided to enhance the detection of phosphothreonine and phosphotyrosine. Although this procedure can be carried out with a protein eluted from a preparative gel and concentrated by trichloroacetic acid or acetone precipitation, it is most easily accomplished by transfer of the protein of interest to a PVDF membrane.

     
 
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 18.2)
  • India ink solution: 1 µl/ml India ink in TBS (unit 18.2)/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
  • 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° 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 10.2), immunoblotting (unit 10.8 or unit 10.19), staining filters (unit 10.7), and autoradiography ( appendix 3A)

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 22)
  • 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