AFM-Based Single-Molecule Force Spectroscopy of Proteins

Methods Mol Biol. 2018:1814:35-47. doi: 10.1007/978-1-4939-8591-3_3.


Single-molecule force spectroscopy by AFM (AFM-SMFS) is an experimental methodology that allows unequivocal sensitivity and control for investigating and manipulating the mechanical properties of single molecules. The past 20 years of AFM-SMFS has provided numerous breakthroughs in the understanding of the mechanical properties and force-induced structural rearrangements of sugars, DNA, and proteins. Here, we focus on the application of AFM-SMFS to study proteins, since AFM-SMFS has succeeded in providing abundant information about protein folding pathways, kinetics, interactions, and misfolding. In this chapter we describe the experimental procedures for conducting a SMFS-AFM experiment-including purification of protein samples, setup and calibration of the AFM instrumentation, and the thorough and unbiased analysis of resulting AFM data.

Keywords: Atomic force spectroscopy; Protein folding; Protein mechanics; Protein misfolding; Single molecule.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Data Analysis
  • Humans
  • Microscopy, Atomic Force / methods*
  • Protein Unfolding
  • Proteins / chemistry*


  • Proteins