This article describes the general method to perform the classical two‐hybrid system. Although it has already been more than 25 years since this technique was developed, it still represents one of the best and most inexpensive, time saving, and straightforward methods to identify and study protein‐protein interactions. Indeed, this system can be easily used to identify interacting proteins for a given protein, to check interactions between two known proteins, or to map interacting domains. Most of the interactions revealed using the two‐hybrid assay have been proven to be binary direct interactions. Data comparison with other systems, such as mass spectrometry, have demonstrated that this system is at least as reliable. In fact, its use is increasing with time, and at present numerous variants of the yeast two‐hybrid assay have been developed, including high‐throughput systems that promote the generation of a proteome‐scale map of protein‐protein interactions in specific system.
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