Single-molecule pull-down (SiMPull) for new-age biochemistry: methodology and biochemical applications of single-molecule pull-down (SiMPull) for probing biomolecular interactions in crude cell extracts

Bioessays. 2014 Nov;36(11):1109-19. doi: 10.1002/bies.201400090. Epub 2014 Aug 29.


Macromolecular interactions play a central role in many biological processes. Protein-protein interactions have mostly been studied by co-immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real-time at the single-molecule level. This technique is called single-molecule pull-down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single-molecule fluorescence microscopy. SiMPull is used to count how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single-molecule biochemical tool to perform functional studies on the pulled-down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools.

Keywords: Western blotting; co-immunoprecipitation; fluorescence; protein complex; pull-down; single molecule.

Publication types

  • Review

MeSH terms

  • Biochemistry
  • Cell Extracts / analysis*
  • Cell Membrane / metabolism
  • Cytosol / metabolism
  • Gene Dosage
  • Microscopy, Fluorescence
  • Multiprotein Complexes / analysis*
  • Multiprotein Complexes / metabolism
  • Protein Interaction Mapping / methods*
  • Protein Interaction Maps


  • Cell Extracts
  • Multiprotein Complexes