Probing cellular protein complexes using single-molecule pull-down.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional ...
pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.
Mesh Terms:
Bacterial Proteins, Cell Extracts, Color, Cyclic AMP-Dependent Protein Kinases, DNA Helicases, Fluorescence Resonance Energy Transfer, Fluorescent Antibody Technique, HEK293 Cells, Humans, Immunoprecipitation, Luminescent Proteins, Microscopy, Fluorescence, Mitochondrial Proteins, Multiprotein Complexes, Photobleaching, Protein Binding, Protein Interaction Mapping, Receptors, Adrenergic, beta, Tissue Extracts
Nature
Date: May. 26, 2011
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