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A fluorescent two-hybrid (F2H) assay for direct visualization of protein interactions in living cells

Item Type:Article
Title:A fluorescent two-hybrid (F2H) assay for direct visualization of protein interactions in living cells
Creators Name:Zolghadr, K. and Mortusewicz, O. and Rothbauer, U. and Kleinhans, R. and Goehler, H. and Wanker, E.E. and Cardoso, M.C. and Leonhardt, H.
Abstract:Genetic high-throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid (F2H) assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the F2H assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real-time.
Keywords:Base Sequence, Cell Cycle, Cell Line, DNA Primers, DNA Repair Enzymes, Green Fluorescent Proteins, Huntington Disease, Lac Operon, Mitochondrial Proteins, Protein Interaction Mapping, Proteomics, Recombinant Fusion Proteins, Repressor Proteins, Transfection, Two-Hybrid System Techniques, Animals, Cricetinae
Source:Molecular and Cellular Proteomics
Publisher:American Society for Biochemistry and Molecular Biology
Page Range:2279-2287
Date:1 November 2008
Official Publication:https://doi.org/10.1074/mcp.M700548-MCP200
PubMed:View item in PubMed

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