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Biophys J. 2018 Jul 17;115(2):283-288. doi: 10.1016/j.bpj.2018.05.013. Epub 2018 May 25.

Toward Single-Cell Single-Molecule Pull-Down.

Author information

1
Department of Physics and Astronomy, Iowa State University, Ames, Iowa; Department of Physics, Center for the Physics of Living Cells and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois.
2
Department of Physics, Center for the Physics of Living Cells and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois.
3
Department of Physics, Center for the Physics of Living Cells and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois; Department of Biochemistry and Biophysics, Center for Phage Technology, Texas A&M University, College Station, Texas.
4
Department of Physics, Center for the Physics of Living Cells and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois; Howard Hughes Medical Institute, Baltimore, Maryland; Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, Maryland; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland. Electronic address: tjha@jhu.edu.

Abstract

Single-molecule pull-down (SiMPull) can capture native protein complexes directly from cell lysates for analysis of complex composition and activities at the single-molecule level. Although SiMPull requires many fewer cells compared to conventional pull-down assays, all studies so far have been performed using lysates from many cells. In principle, extending SiMPull to the single-cell level will allow the investigation of cell-to-cell variations on the stoichiometry and activities of biomolecular complexes. We developed a protocol to lyse bacterial cells in situ and capture the released proteins on the imaging surface using antibodies. The use of lysozymes delayed the protein release until after the flow has ceased, and the use of a 10-μm spacer reduces the capture radius within which ∼70% of target proteins can be captured to below 30 μm. Proteins thus captured can be unambiguously assigned to the originating cell. The developed platform should be compatible with high-throughput protein analysis and protein-protein interaction analysis at the single-cell level through single-molecule imaging.

PMID:
29804751
PMCID:
PMC6050716
[Available on 2019-07-17]
DOI:
10.1016/j.bpj.2018.05.013

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