Tracking unfolding and refolding reactions of single proteins using atomic force microscopy methods

Paul J. Bujalowski, Andres F. Oberhauser

Research output: Contribution to journalArticlepeer-review

18 Scopus citations


During the last two decades single-molecule manipulation techniques such as atomic force microscopy (AFM) has risen to prominence through their unique capacity to provide fundamental information on the structure and function of biomolecules. Here we describe the use of single-molecule AFM to track protein unfolding and refolding pathways, enzymatic catalysis and the effects of osmolytes and chaperones on protein stability and folding. We will outline the principles of operation for two different AFM pulling techniques: length clamp and force-clamp and discuss prominent applications. We provide protocols for the construction of polyproteins which are amenable for AFM experiments, the preparation of different coverslips, choice and calibration of AFM cantilevers. We also discuss the selection criteria for AFM recordings, the calibration of AFM cantilevers, protein sample preparations and analysis of the obtained data.

Original languageEnglish (US)
Pages (from-to)151-160
Number of pages10
Issue number2
StatePublished - Apr 1 2013


  • Atomic force microscopy
  • Biophysics
  • Chaperones
  • Osmolytes
  • Protein folding
  • Single-molecule
  • Unfolding

ASJC Scopus subject areas

  • Molecular Biology
  • General Biochemistry, Genetics and Molecular Biology


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