Probing cellular protein complexes using single-molecule pull-down

Ankur Jain, Ruijie Liu, Biswarathan Ramani, Edwin Arauz, Yuji Ishitsuka, Kaushik Ragunathan, Jeehae Park, Jie Chen, Yang Kevin Xiang, Taekjip Ha

Research output: Contribution to journalArticle

216 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)484-488
Number of pages5
JournalNature
Volume473
Issue number7348
DOIs
StatePublished - May 26 2011
Externally publishedYes

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Proteins
Macromolecular Substances
Tissue Extracts
Cyclic AMP-Dependent Protein Kinases
Fluorescence Microscopy
Organelles
Cytosol
Membranes

ASJC Scopus subject areas

  • General

Cite this

Jain, A., Liu, R., Ramani, B., Arauz, E., Ishitsuka, Y., Ragunathan, K., ... Ha, T. (2011). Probing cellular protein complexes using single-molecule pull-down. Nature, 473(7348), 484-488. https://doi.org/10.1038/nature10016

Probing cellular protein complexes using single-molecule pull-down. / Jain, Ankur; Liu, Ruijie; Ramani, Biswarathan; Arauz, Edwin; Ishitsuka, Yuji; Ragunathan, Kaushik; Park, Jeehae; Chen, Jie; Xiang, Yang Kevin; Ha, Taekjip.

In: Nature, Vol. 473, No. 7348, 26.05.2011, p. 484-488.

Research output: Contribution to journalArticle

Jain, A, Liu, R, Ramani, B, Arauz, E, Ishitsuka, Y, Ragunathan, K, Park, J, Chen, J, Xiang, YK & Ha, T 2011, 'Probing cellular protein complexes using single-molecule pull-down', Nature, vol. 473, no. 7348, pp. 484-488. https://doi.org/10.1038/nature10016
Jain A, Liu R, Ramani B, Arauz E, Ishitsuka Y, Ragunathan K et al. Probing cellular protein complexes using single-molecule pull-down. Nature. 2011 May 26;473(7348):484-488. https://doi.org/10.1038/nature10016
Jain, Ankur ; Liu, Ruijie ; Ramani, Biswarathan ; Arauz, Edwin ; Ishitsuka, Yuji ; Ragunathan, Kaushik ; Park, Jeehae ; Chen, Jie ; Xiang, Yang Kevin ; Ha, Taekjip. / Probing cellular protein complexes using single-molecule pull-down. In: Nature. 2011 ; Vol. 473, No. 7348. pp. 484-488.
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