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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Biophysics
• Single-molecule Assays
• Microscopy
• Molecular Biology
• Molecular/Chemical Probes
• Protein Biochemistry