Single-molecule FRET of protein-nucleic acid and protein-protein complexes: surface passivation and immobilization

Methods. 2010 Oct;52(2):192-200. doi: 10.1016/j.ymeth.2010.06.010. Epub 2010 Jun 8.

Abstract

Single-molecule fluorescence spectroscopy reveals the real time dynamics that occur during biomolecular interactions that would otherwise be hidden by the ensemble average. It also removes the requirement to synchronize reactions, thus providing a very intuitive approach to study kinetics of biological systems. Surface immobilization is commonly used to increase observation times to the minute time scale, but it can be detrimental if the sample interacts non-specifically with the surface. Here, we review detailed protocols to prevent such interactions by passivating the surface or by trapping the molecules inside surface immobilized lipid vesicles. Finally, we discuss recent examples where these methods were applied to study the dynamics of important cellular processes at the single-molecule level.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Review

MeSH terms

  • DNA / chemistry*
  • Fluorescence Resonance Energy Transfer / methods*
  • Lipids / chemistry
  • Nucleic Acid Conformation
  • Polyethylene Glycols / chemistry
  • Protein Structure, Tertiary
  • Proteins / chemistry*
  • RNA / chemistry*

Substances

  • Lipids
  • Proteins
  • Polyethylene Glycols
  • RNA
  • DNA