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

ABSTRACT: 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