Protocol

Protein Acetylation

Volume 981 of the series Methods in Molecular Biology pp 103-113

Date:

Separation and Purification of Multiply Acetylated Proteins Using Cation-Exchange Chromatography

  • Romeo PapazyanAffiliated withDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine
  • , Sean D. TavernaAffiliated withDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine

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Abstract

High-performance liquid chromatography (HPLC) is extremely useful for the study of proteins and the characterization of their posttranslational modifications. Here we describe a method that utilizes cation-exchange HPLC to separate multiply acetylated histone H3 species on the basis of their charge and hydrophilicity. This high-resolution method allows for the separation of histone H3 species that differ by as few as one acetyl group, and is compatible with subsequent analysis by a variety of techniques, including mass spectrometry and western blotting.

Key words

Cation-exchange chromatography PolyCAT A Acetylation HPLC Histone H3 Acetic anhydride