RESUMO
Nucleosomes are the fundamental organizing unit of all eukaryotic genomes. Understanding how proteins gain access to DNA-binding sites located within nucleosomes is important for understanding DNA processing including transcription, replication, and repair. Single-molecule total internal reflection fluorescence (smTIRF) microscopy measurements can provide key insight into how proteins gain and maintain access to DNA sites within nucleosomes. Here, we describe methods for smTIRF experiments including the preparation of fluorophore-labeled nucleosomes, the smTIRF system, data acquisition, analysis, and controls. These methods are presented for investigating transcription factor binding within nucleosomes. However, they are applicable for investigating the binding of any site-specific DNA-binding protein within nucleosomes.