A fluorescence-based protocol to quantitatively titrate CUT&RUN buffer components

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Cleavage under targets and release using nuclease (CUT&RUN) is a key technique for mapping protein-DNA interactions and histone modifications in chromatin.
• Optimizing buffer conditions is crucial for efficient cell permeabilization and maintaining epitope integrity during CUT&RUN assays.

Purpose of the Study:

• To develop and present a fluorescence-based protocol for quantitative titration of CUT&RUN buffer components.
• To optimize cell permeabilization and target epitope retention on chromatin for improved CUT&RUN assay performance.

Main Methods:

• Cells were captured on concanavalin A beads.
• A fluorescence-based assay was employed to titrate digitonin and NaCl concentrations in CUT&RUN buffers.
• Fluorescence imaging was used to determine optimal buffer conditions.

Main Results:

• The developed protocol allows for quantitative assessment of CUT&RUN buffer components.
• Optimal concentrations of digitonin and NaCl were identified to enhance cell permeabilization and epitope retention.
• The fluorescence-based titration provides a robust method for assay optimization.

Conclusions:

• This fluorescence-based titration protocol offers an efficient and quantitative approach to optimize CUT&RUN assays.
• The optimized conditions enhance the reliability and accuracy of identifying genomic binding sites for proteins and histone modifications.