Title: Single Molecule Immuno Pull Down Assay (SiMPull) for Studying Protein-Protein Interactions
Abstract: Protein-protein interactions form the cornerstone for most biological pathways. Governed by numerous factors, same protein can associate with a host of different proteins and exhibit diverse functionality. This heterogeneity in complex composition is difficult to probe using bulk assays like immunoblot. Using TIRF microscopy, we have developed a single molecule immuno pull down assay (SiMPull) enabling direct visualization of protein-protein associations. Using fluorophore labeled antibodies we are able to visualize individual tethered molecules of protein of interest with high specificity. Surface bound antibodies are employed to specifically immobilize a target protein. We are able to pull down protein of interest (bait) from crude cell lysate, eliminating the need for protein purification. The bait protein co-precipitates its interacting partners. The identity of proteins bound to the bait is verified either by using fluorescent protein fusion constructs or through antibodies against anticipated targets. For a multimeric protein complex, fluorophore labeled antibodies against its subunits colocalize in the same diffraction limited spot. Using different dye labels for antibodies against different subunits and multicolor fluorescence colocalization, we are able to ascertain the molecular composition of these complexes. Individual photobleaching events provide us insights about the stoichiometry. SiMPull can be extended to single cell lysate analysis and provides a rapid, sensitive and robust platform for analyzing protein assemblies in situ.