The fluorescent two-hybrid (F2H) assay for direct analysis of protein-protein interactions in living cells.

摘要: Information about protein interactions is crucial for the understanding of cellular processes. Current methods investigation protein-protein (PPIs) require either removal proteins from their normal environment, perturbation cells or costly instrumentation and advanced technical expertise (Fields Song, Nature 340:245-246, 1989; Deane et al., Mol Cell Proteomics 1:349-356, 2002; Kerppola, Nat Rev Biol 7:449-456, 2006; Blanchard 5:2175-2184, Miller 6:1027-1038, 2007; Miyawaki, Dev 4:295-305, 2003; Parrish Curr Opin Biotechnol 17:387-393, Sekar Periasamy, J 160:629-633, 2003). Here, we describe a simple assay to directly visualize analyze PPIs in single living cells. By adapting lac operator/repressor system, generated stable nuclear interaction platform. A fluorescent bait tethered platform assayed co-localization prey fusion proteins. This two-hybrid (F2H) allows cell cycle dependent PPIs. With this based even different subcellular compartments can be visualized real time (Zolghadr 7:2279-2287, 2008). The optical readout enables automated imaging systems segment acquired data high-throughput screening response external stimuli chemical compounds.