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Protocol to prepare doubly labeled fluorescent nucleosomes for single-molecule fluorescence microscopy

ABSTRACT: Summary Single-molecule fluorescence microscopy (SMFM) has been shown to be informative in understanding the interaction of chromatin-associated factors with nucleosomes, the basic building unit of chromatin. Here, we present a protocol for preparing doubly labeled fluorescent nucleosomes for SMFM. We describe steps for over-expression in E. coli and purification of recombinant human core histones. We then detail fluorescent labeling of histones and nucleosomal double-stranded DNA followed by octamer refolding and nucleosome reconstitution. Graphical abstract Highlights • Over-expression in E. coli and purification of recombinant human core histones• Site-specific fluorescent labeling of histones• PCR-based site-specific labeling of nucleosomal DNA• Refolding of fluorescent octamer and reconstitution of fluorescent nucleosome Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Single-molecule fluorescence microscopy (SMFM) has been shown to be informative in understanding the interaction of chromatin-associated factors with nucleosomes, the basic building unit of chromatin. Here, we present a protocol for preparing doubly labeled fluorescent nucleosomes for SMFM. We describe steps for over-expression in E. coli and purification of recombinant human core histones. We then detail fluorescent labeling of histones and nucleosomal double-stranded DNA followed by octamer refolding and nucleosome reconstitution.

SUBMITTER: Ghoneim M

PROVIDER: S-EPMC10148226 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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