Journal
NATURE
Volume 473, Issue 7348, Pages 484-U322Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nature10016
Keywords
-
Categories
Funding
- NIH [AI083025, GM065367, HL082846, AR048914]
- NSF [0646550, 0822613]
- Division Of Physics
- Direct For Mathematical & Physical Scien [0646550] Funding Source: National Science Foundation
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available