Probing cellular protein complexes using single-molecule pull-down

single-molecule;fluorescence microscopy;protein interactions

Jain, Ankur

关键词: single-molecule;    fluorescence microscopy;    protein interactions;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/46887/Ankur_Jain.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Cellular processes result from dynamic interactions between biomolecules. The gold standard method for investigating interaction between biomolecules is the pull-down assay. We have extended the conventional pull-down assay to its ultimate limit: the analysis of single biomolecular complexes. We achieve this goal by performing the pull-downs directly on to the surface of microscope slides and visualizing the captured biomolecules at single-molecule resolution using total internal reflection fluorescence (TIRF) microscopy. We name this technology single-molecule pull-down or SiMPull. In one reification biotinylated antibody against the protein of interest is immobilized on a flow chamber. The flow chambers are passivated using a polymer coating such that the cellular components do not bind, and the immobilized antibody specifically captures the target protein. Cell lysates are diluted to obtain isolated molecules suitable for single-molecule imaging. The target molecules are fluorescently labeled either using a genetically encoded fluorescent protein tag or using antibodies, and imaged using a single-molecule TIRF microscope. Using SiMPull we are able to discriminate between multiple association states of a protein as well as determine the stoichiometry of interaction. This technology is widely applicable to an array of biological contexts, and is suitable to analysis of endogenous protein complexes from animal tissue. In particular, we have used SiMPull to investigate the architecture and assembly of mechanistic target of rapamycin complexes. The complexes captured from cell extracts on to our imaging chambers retain their functional activities: thus SiMPull can be used as a preparatory tool for single-molecule biochemical analysis on proteins that cannot be readily purified or reconstituted. Finally, we have extended this approach to the analysis of lipid-protein interactions.