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The Nucleus pp 285-302 | Cite as

Purification and Analysis of Variant and Modified Histones Using 2D PAGE

  • George R. Green
  • Duc P. Do
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 464)

Abstract

Two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) systems employing combinations of acetic acid/urea (AU), acetic acid/urea/Triton X-100 (AUT) and sodium dodecyl sulfate (SDS) gel formulations are uniquely effective for resolution of histone variants and their modified derivatives. Coupled with Western transfer methods using modification-specific antibodies and recent advances in mass spectrometry, 2D PAGE emerges as a versatile tool for histone purification and analysis. This chapter describes 2D PAGE gel systems appropriate for histone proteins, including detailed procedures for designing, running, and staining gels. Methods for electrophoretic transfer of histones from AUT×SDS and AUT×AU 2D gels are described and evaluated. Alternatively, methods are provided for obtaining highly purified protein samples from fixed and stained gels via electroelution of proteins from specific gel spots.

Keywords

Histone variants Histone modification Phosphorylation Acetylation Ubiquitylation Two-dimensional polyacrylamidegelelectrophoresis Electroelution Western analysis Triton X-100 SDS-PAGE 

Notes

Acknowledgments

We thank Eric Selker, Kristina Smith, and Keyur Adhvaryu for providing Neurospora strains and assistance with isolating nuclei and histones from this species.

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Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • George R. Green
    • 1
  • Duc P. Do
    • 1
  1. 1.Department of Pharmaceutical SciencesMercer University College of Pharmacy and Health SciencesAtlantaUSA

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