Toward Single-Cell Single-Molecule Pull-Down

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.