Abstract
Due to its ability to track distance changes within individual molecules or molecular complexes on the nanometer scale and in real time, single-molecule fluorescence resonance energy transfer (single-molecule FRET) is a powerful tool to tackle a wide range of important biological questions. Using our recently developed single-molecule FRET assay to monitor nucleosome translocation as an illustrative example, we describe here in detail how to set up, carry out, and analyze single-molecule FRET experiments that provide time-dependent information on biomolecular processes.