Separation and Purification of Multiply Acetylated Proteins Using Cation-Exchange Chromatography
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 wordsCation-exchange chromatography PolyCAT A Acetylation HPLC Histone H3 Acetic anhydride
We thank T. Gilbert and other members of the Taverna laboratory for comments on this protocol. This work was supported by NIH grant RO190035489.
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