A Novel Nanobead-Based Single-Molecule Pull-Down For Cell Populations and Single Cells

Abstract

Conventional co-immunoprecipitation methods (co-IP/pull-down) commonly used for accessing protein-protein interactions (PPIs) require large numbers of cells and are thus unsuitable for studying PPIs in rare cells such as sensory hair cells, circulating tumor cells, and embryonic stem cells; this substantially hampers rare-cell research and therefore calls for the development of single-cell pull-down techniques. Equally important, such single-cell pull-down techniques enable investigation of cell-to-cell variation in PPIs. Recently, based on the principle of a published single-molecule pull-down assay (SiMPull), two single-cell pull-down techniques were developed. However, both methods present a considerably high technical barrier and can be applied only to very limited cell types such as bacteria or adherent cells and not to primary- or suspension-culture cells. Here, we report a highly innovative nanobead-based approach for SiMPull designed for cell populations and, more importantly, single cells. We used commercially available, pre-surface-functionalized magnetic nanobeads to capture the protein of interest together with its binding partners specifically from cell extracts and observed these interactions by fused fluorescent proteins or immunofluorescent labeling under microscope. For the single-cell SiMPull, we used microwell array chips to trap single cells and applied magnetic nanobeads to microwells. After cell lysis, the nanobeads captured target proteins and pulled them down to the glass bottom of the microwell upon imposing a magnetic field, followed by immunofluorescent labeling and visualization under microscope. Our methods are substantially simpler and faster than existing SiMPull techniques for cell populations and single cells and are considerably more widely applicable to all cell types. These two crucial features would enable universal application of our methods in common biological and clinical laboratories. The work was supported by Hong Kong RGC Grant GRF16111616.