Using the Yeast Two-Hybrid System to Identify Interacting Proteins

  • John Miller
  • Igor Stagljar
Part of the Methods in Molecular Biology book series (MIMB, volume 261)


The yeast two-hybrid system is a powerful technique for studying protein-protein interactions. Two proteins are separately fused to the independent DNA-binding and transcriptional activation domains of the Gal4p transcription factor. If the proteins interact, they reconstitute a functional Gal4p that activates expression of reporter gene(s). In this way, two individual proteins may be tested for their ability to interact, and a transcriptional readout can be measured to detect this interaction. Furthermore, novel interacting partners can be found by screening a single protein or domain against a library of other proteins using this system. It is this latter feature—the ability to search for interacting proteins without any prior knowledge of the identity of such proteins—that is the most powerful application of the two-hybrid technique.

Key Words

Two-hybrid methodology protocol library screening 


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Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • John Miller
    • 1
  • Igor Stagljar
    • 2
  1. 1.Department of Genome SciencesUniversity of WashingtonSeattle
  2. 2.Institute of Veterinary Biochemistry and Molecular BiologyUniversity of Zurich-IrchelZurichSwitzerland

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