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Updated: Jul 12, 2025

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions
Published on: February 1, 2010
Protocol for establishing a protein interactome based on close physical proximity to a target protein within live
Janhavi A Kolhe1, Neethu L Babu1, Brian C Freeman1
1Department of Cell and Developmental Biology, School of Molecular and Cellular Biology, University of Illinois-Urbana-Champaign, Urbana, IL, USA.
Abstract:
Here, we present a protocol for establishing a protein interactome based on close physical proximity to a target protein within live yeast cells. We describe steps for capturing both transient and stable binders by integrating a non-natural amino acid. We detail procedures for employing a site-directed method for labeling the surface that mediates protein associations and uncovers the binding sites on the interactors. Combined with mass spectrometry, our approach proves valuable in discovering binding partners and constructing a comprehensive protein-interaction network.
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