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Probing cellular protein complexes using single-molecule pull-down

Author

Listed:
  • Ankur Jain

    (Center for Biophysics and Computational Biology and Institute for Genomic Biology, University of Illinois at Urbana-Champaign)

  • Ruijie Liu

    (University of Illinois at Urbana-Champaign)

  • Biswarathan Ramani

    (University of Illinois at Urbana-Champaign)

  • Edwin Arauz

    (University of Illinois at Urbana-Champaign)

  • Yuji Ishitsuka

    (University of Illinois at Urbana-Champaign
    Howard Hughes Medical Institute)

  • Kaushik Ragunathan

    (Center for Biophysics and Computational Biology and Institute for Genomic Biology, University of Illinois at Urbana-Champaign)

  • Jeehae Park

    (Center for Biophysics and Computational Biology and Institute for Genomic Biology, University of Illinois at Urbana-Champaign)

  • Jie Chen

    (University of Illinois at Urbana-Champaign)

  • Yang K. Xiang

    (University of Illinois at Urbana-Champaign)

  • Taekjip Ha

    (Center for Biophysics and Computational Biology and Institute for Genomic Biology, University of Illinois at Urbana-Champaign
    University of Illinois at Urbana-Champaign
    Howard Hughes Medical Institute)

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.

Suggested Citation

  • Ankur Jain & Ruijie Liu & Biswarathan Ramani & Edwin Arauz & Yuji Ishitsuka & Kaushik Ragunathan & Jeehae Park & Jie Chen & Yang K. Xiang & Taekjip Ha, 2011. "Probing cellular protein complexes using single-molecule pull-down," Nature, Nature, vol. 473(7348), pages 484-488, May.
    • Handle: RePEc:nat:nature:v:473:y:2011:i:7348:d:10.1038_nature10016
    DOI: 10.1038/nature10016
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    Cited by:

    1. George K. E. Umanah & Leire Abalde-Atristain & Mohammed Repon Khan & Jaba Mitra & Mohamad Aasif Dar & Melissa Chang & Kavya Tangella & Amy McNamara & Samuel Bennett & Rong Chen & Vasudha Aggarwal & Ma, 2022. "AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1," Nature Communications, Nature, vol. 13(1), pages 1-18, December.
    2. Zengjie Xia & Emily E. Prescott & Agnieszka Urbanek & Hollie E. Wareing & Marianne C. King & Anna Olerinyova & Helen Dakin & Tom Leah & Katy A. Barnes & Martyna M. Matuszyk & Eleni Dimou & Eric Hidari, 2024. "Co-aggregation with Apolipoprotein E modulates the function of Amyloid-β in Alzheimer’s disease," Nature Communications, Nature, vol. 15(1), pages 1-18, December.

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