In Situ Quantitative Imaging of Single-Molecule Co-Immunoprecipitation

Co-immunoprecipitation (co-IP) and western blotting are the gold standards for assessing protein-protein interactions. However, their protein band output only provides qualitative and static information on protein compositions and their interactions. In this study, we demonstrate a quantitative transformation of the traditional western and co-ip techniques using in situ single-molecule level measurements with millisecond time resolution. We have quantified the molar concentration of specific proteins in whole cell or tissue extracts and determined the stoichiometry and kinetics of their interactions. We have then used our methods to quantify the expression level and signaling frequency of oncogenic Ras that has been directly pulled down from tumor tissues. Our results suggest that these techniques will make valuable additions to the molecular diagnostic repertoire of modern biomedicine.Remark to the program chairsRecently, Dr. Taekjip Ha and co-workers reported on single-molecule pull down. They showed that target proteins of tiny amount can be selectively immobilized on surface while thousands of other proteins are effectively rejected from the same surface (Nature 473, 484 (2011)). Inspired by this approach, we have transformed the co-IP and western blotting into quantitative methods. By employing fluorescently labeled binding competitors, we demonstrate single-molecule western analysis, in which we robustly measure the true molar concentration of specific proteins in cell or tissue extracts. We also demonstrate that in situ imaging can be done during single-molecule co-IP, which crucially enables to access weak and transient protein-protein interactions. Finally, we show that oncogenic Ras proteins pulled down from a tumor tissue indeed shows a higher activation percentage as well as a higher expression level than Ras from normal tissues. Our techniques rapidly assess the strength of a certain signaling step, which should be useful for the molecular diagnostics.