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Nat Protoc. 2014 Sep;9(9):2090-9. doi: 10.1038/nprot.2014.142. Epub 2014 Aug 7.

Chromatin enrichment for proteomics.

Author information

1
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
2
1] Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK. [2] Department of Biotechnology, Institute of Bioanalytics, Technische Universität Berlin, Berlin, Germany.

Abstract

During interphase, chromatin hosts fundamental cellular processes, such as gene expression, DNA replication and DNA damage repair. To analyze chromatin on a proteomic scale, we have developed chromatin enrichment for proteomics (ChEP), which is a simple biochemical procedure that enriches interphase chromatin in all its complexity. It enables researchers to take a 'snapshot' of chromatin and to isolate and identify even transiently bound factors. In ChEP, cells are fixed with formaldehyde; subsequently, DNA together with all cross-linked proteins is isolated by centrifugation under denaturing conditions. This approach enables the analysis of global chromatin composition and its changes, which is in contrast with existing chromatin enrichment procedures, which either focus on specific chromatin loci (e.g., affinity purification) or are limited in specificity, such as the analysis of the chromatin pellet (i.e., analysis of all insoluble nuclear material). ChEP takes half a day to complete and requires no specialized laboratory skills or equipment. ChEP enables the characterization of chromatin response to drug treatment or physiological processes. Beyond proteomics, ChEP may preclear chromatin for chromatin immunoprecipitation (ChIP) analyses.

PMID:
25101823
PMCID:
PMC4300392
DOI:
10.1038/nprot.2014.142
[Indexed for MEDLINE]
Free PMC Article

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