Single Molecule FRET Analysis of DNA Binding Proteins

  • Kathy R. ChaurasiyaEmail author
  • Remus T. Dame
Part of the Methods in Molecular Biology book series (MIMB, volume 1665)


The complex binding dynamics between DNA and proteins are often obscured by ensemble averaging effects in conventional biochemical experiments. Single-molecule fluorescence methods are powerful tools to investigate DNA–protein interaction dynamics in real time. In this chapter, we focus on using single-molecule Förster Resonance Energy Transfer (smFRET) to probe the binding dynamics of individual proteins on single DNA molecules. We provide a detailed discussion of total internal reflection fluorescence (TIRF) instrument design, nucleic acid labeling with fluorophores, flow cell surface passivation, and data analysis methods.

Key words

Single molecule FRET TIRF Nucleic acid–protein interaction DNA binding protein PEG surface passivation 



The authors thank all the members of the David Rueda laboratory for training in single-molecule FRET methods. This work was supported in part by the Human Frontiers Science Program [RGP0014/2014] and the Netherlands Organization for Scientific Research [VICI 016.160.613].


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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Leiden Institute of ChemistryLeiden UniversityLeidenThe Netherlands

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