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

Mol Cell Proteomics. 2008 Nov;7(11):2279-87. doi: 10.1074/mcp.M700548-MCP200. Epub 2008 Jul 12.


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 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 fluorescent two-hybrid 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Cycle
  • Cell Line
  • Cricetinae
  • DNA Primers / genetics
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Huntington Disease / genetics
  • Huntington Disease / metabolism
  • Lac Operon
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism
  • Protein Interaction Mapping / methods*
  • Proteomics / methods
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Transfection
  • Two-Hybrid System Techniques*


  • DNA Primers
  • Mitochondrial Proteins
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Green Fluorescent Proteins
  • DNA Repair Enzymes