Sm-ChIPi: Single-Molecule Chromatin Immunoprecipitation Imaging

  • Roubina Tatavosian
  • Xiaojun RenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1689)


Epigenetic complexes regulate chromatin dynamics via binding to and assembling on chromatin. However, the mechanisms of chromatin binding and assembly of epigenetic complexes within cells remain incompletely understood, partly due to technical challenges. Here, we present a new approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) that enables to assess the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was developed based on chromatin immunoprecipitation followed by single-molecule fluorescence microscopy imaging. In this method, an epigenetic complex subunit fused with a gene coding for a fluorescent protein is stably expressed in its corresponding knockout cells. Nucleosomes associated with epigenetic complexes are isolated from cells at native conditions and incubated with biotinylated antibodies. The resulting complexes are immobilized on a quartz slide that had been passivated and functionalized with NeutrAvidin. Image stacks are then acquired by using single-molecule TIRF microscopy. The individual spots imaged by TIRF microscopy represent single protein–nucleosome complexes. The number of copies of the protein complexes on a nucleosome is inferred from the fluorescence photobleaching measurements. Sm-ChIPi is a sensitive and direct method that can quantify the cellular assembly stoichiometry of epigenetic complexes on chromatin.

Key words

Epigenetics Epigenetic complex Chromatin Polycomb group (PcG) proteins PRC2 PRC1 Nucleosome Chromatin-binding mechanism Epigenetic protein–nucleosome complex Single-molecule fluorescent microscopy 



This work was supported by the National Cancer Institute of the National Institutes of Health under Award Number R03CA191443 (to X.R.).


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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Colorado DenverDenverUSA
  2. 2.Department of ChemistryUniversity of Colorado DenverDenverUSA

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