1. Sequential Chromatin Immunoprecipitation to Identify Heterotypic Nucleosomes
- Author
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Maxim Nekrasov and David J. Tremethick
- Subjects
Affinity purified antibody ,education.field_of_study ,animal structures ,biology ,Chemistry ,Immunoprecipitation ,Population ,Chromatin ,Cell biology ,Histone ,embryonic structures ,biology.protein ,Nucleosome ,education ,Chromatin immunoprecipitation ,Micrococcal nuclease - Abstract
Sequential ChIP (ChIP-reChIP) enables the characterization of the same nucleosome for two different types of modifications or histone subtypes. Here, we describe a ChIP-reChIP protocol to identify a heterotypic (asymmetric) H2A.Z-H2A-containing nucleosome. In this method, following MNase digestion of chromatin to mostly a mononucleosome fraction, H2A.Z-containing nucleosomes are first immunoprecipitated using affinity purified anti-H2A.Z antibodies. This H2A.Z-containing nucleosome fraction is then subsequently immunoprecipitated using anti-H2A affinity purified antibodies to yield an enriched population of heterotypic H2A.Z-H2A containing nucleosomes. This protocol can be adopted to investigate any pair-wise combination of any histone variant, histone posttranslational modification, or any other protein that binds to a modified nucleosome.
- Published
- 2021