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Extraction, purification and analysis of histones

Abstract

Histone proteins are the major protein components of chromatin, the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. Chromatin is the substrate of many biological processes, such as gene regulation and transcription, replication, mitosis and apoptosis. Since histones are extensively post-translationally modified, the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Many different biochemical techniques have been developed to purify and separate histone proteins. Here, we present standard protocols for acid extraction and salt extraction of histones from chromatin; separation of extracted histones by reversed-phase HPLC; analysis of histones and their specific post-translational modification profiles by acid urea (AU) gel electrophoresis and the additional separation of non-canonical histone variants by triton AU(TAU) and 2D TAU electrophoresis; and immunoblotting of isolated histone proteins with modification-specific antibodies.

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Figure 1: Depiction of histones in the nucleosome.
Figure 2: Schematic flowchart of histone isolation and purification procedures.
Figure 3: Coomassie Blue–stained SDS gel with acid- and salt-extracted histones.
Figure 4: Chromatogram and Coomassie-stained gels from reversed-phase HPLC (RP-HPLC) separation of histones.
Figure 5: Assembly of a 2D triton acid urea (TAU) gel.
Figure 6: Examples of triton acid urea (TAU), AU and 2D TAU gels.

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Acknowledgements

We are grateful to all the previous and current members of the Allis lab who contributed to the development of these techniques. D.S. is the recipient of a fellowship from the Irma T. Hirschl Trust, H.L.D. is supported by a predoctoral fellowship from the Boehringer Ingelheim Foundation and S.B.H. is supported by the Deutsche Forschungsgemeinschaft (DFG).

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Correspondence to Sandra B Hake.

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Shechter, D., Dormann, H., Allis, C. et al. Extraction, purification and analysis of histones. Nat Protoc 2, 1445–1457 (2007). https://doi.org/10.1038/nprot.2007.202

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