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

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.

Author information

1
Center for Biophysics and Computational Biology, University of Illinois Urbana Champaign, Urbana, IL, USA.

Abstract

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

PMID:
25168682
DOI:
10.1002/bies.201400090
[Indexed for MEDLINE]

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