Fig: Real-time detection of protein interactions in cells on the example of the Nrf2-Keap1 interaction
Cell signaling is a highly flexible and transient process that relies on a careful balance between inhibitory and activating signals mediated by protein–protein interactions. A variety of methods for monitoring cellular protein–protein interactions have been described that suffer enormous limitations to capture those highly dynamic signaling events in cells. (i) Ectopic, usually transient overexpression risks non-biologically relevant interactions or artificial modulation of the signaling event itself by disturbing the stoichiometric balance. (ii) Analyzing the protein-protein interactions or signaling state of a cell at a specific time point in end-point assays can lead to misinterpretation or oversight of important underlying processes. (iii) Methods relying on the detection of endogenous protein require either high amounts of protein or special instrumentation, making them incompatible with high-throughput applications. Here present two ultra-sensitive methods—RT-bind and EndoBind—that overcome these limitations and are capable of capturing real-time cellular protein–protein interactions and dynamics of exogenously (RT-bind) or endogenously (EndoBind) expressed proteins in high-throughput.
Bill, A., Espinola, S., Guthy, D. et al. EndoBind detects endogenous protein-protein interactions in real time. Commun Biol 4, 1085 (2021). https://doi.org/10.1038/s42003-021-02600-5