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

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The Nucleus

Part of the book series: Methods in Molecular Biology ((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.

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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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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Mercer University College of Pharmacy and Health Sciences, Atlanta, GA, USA

    George R. Green & Duc P. Do

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  1. George R. Green
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  2. Duc P. Do
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Editors and Affiliations

  1. Hôtel-Dieu Hospital, Laval University Cancer Research Centre, Québec, Canada

    Ronald Hancock

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

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Green, G.R., Do, D.P. (2008). Purification and Analysis of Variant and Modified Histones Using 2D PAGE. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 464. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-461-6_16

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  • DOI: https://doi.org/10.1007/978-1-60327-461-6_16

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-460-9

  • Online ISBN: 978-1-60327-461-6

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