title: 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.
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.
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois Urbana Champaign, Urbana, IL, USA.
authors.forename: Vasudha
authors.initials: V
authors.lastname: Aggarwal
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D001671:Biochemistry; D002457:Cell Extracts / Q000032:analysis*; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018628:Gene Dosage; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000032:analysis* / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps
chemical_list: D002457:Cell Extracts; D046912:Multiprotein Complexes
keywords: Western blotting; co-immunoprecipitation; fluorescence; protein complex; pull-down; single molecule