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

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

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1
Munich Center for Integrated Protein Science (CiPSM) and Department of Biology, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.

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 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.

PMID:
18622019
DOI:
10.1074/mcp.M700548-MCP200
[Indexed for MEDLINE]
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