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

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Single-molecule fluorescence microscopy (SMFM) is crucial for studying chromatin-associated factor interactions.
• Nucleosomes, the fundamental units of chromatin, are key targets for understanding gene regulation.
• Advanced techniques are needed to visualize these interactions at the molecular level.

Purpose of the Study:

• To present a detailed protocol for preparing doubly labeled fluorescent nucleosomes.
• To enable high-resolution SMFM studies of nucleosome dynamics and interactions.
• To provide a reproducible method for researchers in molecular biology and biophysics.

Main Methods:

• Over-expression and purification of recombinant human core histones in E. coli.
• Fluorescent labeling of both histone proteins and nucleosomal DNA.
• Octamer refolding and subsequent nucleosome reconstitution.

Main Results:

• A robust protocol for generating doubly labeled fluorescent nucleosomes.
• Demonstration of the applicability of these nucleosomes in SMFM.
• Successful reconstitution of functional nucleosome structures.

Conclusions:

• The developed protocol facilitates the preparation of essential tools for advanced chromatin research.
• This method enhances the capability of SMFM to probe nucleosome-protein interactions.
• The findings offer a valuable resource for the molecular biology community studying chromatin structure and function.