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Probing cellular protein complexes using single-molecule pull-down

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.

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Figure 1: Schematic for SiMPull assay.
Figure 2: PKA pull-down.
Figure 3: Applications of SiMPull assay.
Figure 4: PcrA pull-down and activity.

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Acknowledgements

We thank S. Myong, P. Jena, S. Arslan and R. Vafabakhsh for discussions. The expression vector encoding the YFP-MAVS gene was a gift from D. Lamarre. This work was funded by NIH grants (AI083025, GM065367 to T.H.; HL082846 to Y.K.X.; AR048914 to J.C.). Additional support was provided by NSF grants (0646550, 0822613 to T.H.). T.H. is an investigator with the Howard Hughes Medical Institute.

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Authors

Contributions

A.J., Y.K.X. and T.H. designed the research. A.J., R.L. and Y.I. conducted experiments, R.L., B.R., E.A., J.C. and J.P. provided samples, K.R. and Y.I. contributed important ideas to the experiments, A.J. and R.L. analysed the data and A.J., Y.K.X. and T.H. wrote the paper with inputs from other authors.

Corresponding authors

Correspondence to Yang K. Xiang or Taekjip Ha.

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The authors declare no competing financial interests.

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Jain, A., Liu, R., Ramani, B. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011). https://doi.org/10.1038/nature10016

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