Separation of Histone Variants and Post‐Translationally Modified Isoforms by Triton/Acetic Acid/Urea Polyacrylamide Gel Electrophoresis

Colleen A. Ryan1, Anthony T. Annunziato1

1 Boston College, Chestnut Hill, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.2
DOI:  10.1002/0471142727.mb2102s45
Online Posting Date:  May, 2001
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Abstract

Due to their similarities in size and charge, complete resolution of histones by electrophoresis poses a considerable challenge. The addition of nonionic detergents to the traditional acetic acid/urea (AU) polyacrylamide gel electrophoresis (PAGE) system has afforded an excellent method to separate not only the different modified forms of histones, but also the primary sequence variant subtypes of selected histone species; it is widely used to separate histones with varying levels of acetylation. This unit describes the use of gels containing the nonionic detergent Triton X‐100, referred to as Triton/acetic acid/urea (TAU) polyacrylamide gels, for analysis of histones. Also included are support protocols detailing several accessory techniques: assembly of gel plates for the TAU gel, preparation of histones from isolated nuclei in a solubilized form amenable to electrophoresis, and electrophoretic transfer of proteins from these gels to PVDF membranes.

     
 
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Table of Contents

  • Basic Protocol 1: Triton/Acetic Acid/Urea (TAU) Polyacrylamide Gel Electrophoresis for Analysis of Histones
  • Support Protocol 1: Assembly of Gel Plates
  • Support Protocol 2: Histone Isolation from Prepared Nuclei
  • Support Protocol 3: Electrophoretic Transfer of TAU‐Polyacrylamide Gels
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Triton/Acetic Acid/Urea (TAU) Polyacrylamide Gel Electrophoresis for Analysis of Histones

  Materials
  • Glacial acetic acid (17.4 M)
  • TEMED (N,N,N′,N′‐tetramethylethylenediamine)
  • recipe9.5 M urea (see recipe)
  • 10% (w/v) ammonium persulfate (APS; prepare fresh for each gel)
  • 10% Triton X‐100 (protein grade; Calbiochem)
  • recipe63.5% acrylamide/0.4% bisacrylamide (see recipe)
  • Moderately warm water (∼45°C) in a beaker
  • recipeRunning buffer (0.9 M acetic acid; see recipe)
  • recipeScavenger solution (2‐mercaptoethylamine; see recipe)
  • 1000‐ and 100‐ml sidearm flasks
  • Gel‐casting assembly (see protocol 2)
  • Vertical gel electrophoresis apparatus to accommodate 16 × 22–cm glass plates (see protocol 2)
  • Power supply capable of running in constant current mode, with two leads
  • 1.5‐mm‐thick 16‐well gel comb
  • Disposable syringes with 23‐G needles
  • Disposable syringe with 18‐G needle bent into a U shape
  • Hamilton syringe
  • Additional reagents and equipment for degassing gel solutions (unit 10.2)

Support Protocol 1: Assembly of Gel Plates

  Materials
  • 95% ethanol
  • 1% (w/v) agarose, melted
  • 16 × 22–cm notched glass plate
  • 16 × 22–cm glass plate
  • Two 22‐cm‐long × 1.5‐mm‐thick Teflon spacers
  • 18‐cm‐long × 1.5‐mm‐thick Teflon spacer
  • Six 5‐cm‐wide stainless steel binder clips (Research Products International)
  • Resealable plastic bag large enough to contain gel‐casting assembly

Support Protocol 2: Histone Isolation from Prepared Nuclei

  Materials
  • Isolated nuclei (e.g., see unit 12.1)
  • Concentrated sulfuric acid (H 2SO 4; 18 M H 2SO 4) diluted to 0.2 M
  • 100% trichloroacetic acid (TCA)
  • 11.6 M hydrochloric acid (HCl)
  • 100% acetone
  • recipeAcetic acid/urea sample buffer (see recipe)

Support Protocol 3: Electrophoretic Transfer of TAU‐Polyacrylamide Gels

  Materials
  • recipeWash buffers I and recipeII (see reciperecipes)
  • recipeCAPS transfer buffer (see recipe)
  • 100% methanol
  • Ponceau S stain (Sigma; optional)
  • Transfer membrane (e.g., Immobilon‐P PVDF membrane, Millipore)
  • Filter paper cut to size of gel
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Figures

Videos

Literature Cited

   Delcuve, G.P. and Davie, J.R. 1992. Western blotting and immunochemical detection of histones electrophoretically resolved on acid‐urea‐triton and sodium dodecyl sulfate‐polyacrylamide gels. Anal. Biochem. 200:339‐341
   Franklin, S.G. and Zweidler, A. 1977. Non‐allelic variants of histones 2a, 2b, and 3 in mammals. Nature 266:273‐275
   Knehr, M., Poppe, M., Enulescu, M., Eickelbaum, W., Stoehr, M., Schroeter, D. and Paweletz, N. 1995. A critical appraisal of synchronization methods applied to achieve maximal enrichment of HeLa cells in specific cell cycle phases. Exp. Cell Res. 217:546‐553
   Mizzen, C.A. and Allis, C.D. 1998. Linking histone acetylation to transcriptional regulation. Cell. Mol. Life Sci. 54:6‐20.
   Panyim, S. and Chalkley, R. 1969. High resolution acrylamide gel electrophoresis of histones. Arch. Biochem. Biophys. 130:337‐346
   Peterson, D.F. and Anderson, E.C. 1964. Quantity production of synchronized mammalian cells in suspension culture. Nature 203:642‐643
   van Holde, K.E. 1988. Chromatin. Springer‐Verlag, New York.
   Waterborg, J.H. and Harrington, R.E. 1987. Western blotting of histones from acid‐urea‐triton‐ and sodium dodecyl sulfate‐polyacrylamide gels. Anal. Biochem. 162:430‐434
   Wolffe, A.P. 1995. Chromatin. Academic Press, San Diego.
   Zweidler, A. 1978. Resolution of histones by polyacrylamide gel electrophoresis in presence of nonionic detergents. Methods Cell Biol. 17:223‐233
Key References
   Zweidler, 1978. See above.
  Initial description of Triton/acetic acid/urea polyacrylamide gel electrophoresis.
   Delcuve and Davie, 1978. See above.
  Description of the development of a method for efficiently transferring the Triton/acetic acid/urea polyacrylamide gels to membrane.
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