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  High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays

Fiebitz, A., Nyarsik, L., Haendler, B., Hu, Y.-H., Wagner, F., Thamm, S., et al. (2008). High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays. BMC Genomics, 9, 68-68. doi:10.1186/1471-2164-9-68.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-806C-C Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-806D-A
Genre: Journal Article

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 Creators:
Fiebitz, Andrea1, Author              
Nyarsik, Lajos2, Author
Haendler, Bernard, Author
Hu, Yu-Hui3, Author              
Wagner, Florian, Author
Thamm, Sabine4, Author              
Lehrach, Hans1, Author              
Janitz, Michal1, Author              
Vanhecke, Dominique2, Author
Affiliations:
1Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433550              
2Max Planck Society, ou_persistent13              
3Dept. of Human Molecular Genetics (Head: Hans-Hilger Ropers), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433549              
4Human Chromosome 21 (Marie-Laure Yaspo), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1479652              

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 Abstract: Background Most of the biological processes rely on the formation of protein complexes. Investigation of protein-protein interactions (PPI) is therefore essential for understanding of cellular functions. It is advantageous to perform mammalian PPI analysis in mammalian cells because the expressed proteins can then be subjected to essential post-translational modifications. Until now mammalian two-hybrid assays have been performed on individual gene scale. We here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of DNA microarrays. Results In this cell array protein-protein interaction assay (CAPPIA), mixtures of bait and prey expression plasmids together with an auto-fluorescent reporter are immobilized on glass slides in defined array formats. Adherent cells that grow on top of the micro-array will become fluorescent only if the expressed proteins interact and subsequently trans-activate the reporter. Using known interaction partners and by screening 160 different combinations of prey and bait proteins associated with the human androgen receptor we demonstrate that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. Moreover, different strategies in respect to bait-prey combinations are presented. Conclusion We demonstrate that the CAPPIA assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. The high number of preys that can be tested per slide together with the flexibility to interrogate any bait of interest and the small amounts of reagents that are required makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.

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Language(s): eng - English
 Dates: 2008-02-06
 Publication Status: Published in print
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Title: BMC Genomics
Source Genre: Journal
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Pages: - Volume / Issue: 9 Sequence Number: - Start / End Page: 68 - 68 Identifier: ISSN: 1471-2164)