Detection of Protein-Protein Interactions Utilizing the Split-Ubiquitin Membrane-Based Yeast Two-Hybrid System.

Siddhartha Dutta, Matthew D Smith

Methods in molecular biology (Clifton, N.J.) 2023

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Identifying the interactors of a protein is a key step in understanding its possible cellular function(s). Among the various methods that can be used to study protein-protein interactions (PPIs), the yeast two-hybrid (Y2H) assay is one of the most standardized, sensitive, and cost-effective in vivo methods available. The most commonly used GAL4-based Y2H system utilizes the yeast transcription factor GAL4 to detect interactions between soluble proteins. By virtue of involving a transcription factor, the protein-protein interactions occur in the nucleus. The split-ubiquitin Y2H system offers an alternative to the traditional GAL4-based Y2H system and takes advantage of the reconstitution of split-ubiquitin in the cytosol to identify interactions between two proteins. Moreover, new membranous and soluble interacting partner(s) can be identified by screening a target protein against proteins produced from a cDNA library using this system. © 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Citation

Siddhartha Dutta, Matthew D Smith. Detection of Protein-Protein Interactions Utilizing the Split-Ubiquitin Membrane-Based Yeast Two-Hybrid System. Methods in molecular biology (Clifton, N.J.). 2023;2690:37-57