Steps for detecting Protein-protein interaction.

1.  A bait vector is created consisting of selected protein to act as a bait (protein of interest against which interaction is to be checked) and DNA binding domain. The product of this vector is a Fusion protein consisting of bait protein and DNA binding domain.

2.  Another Prey vector is created consisting of transcription activation domain and a specific protein whose interaction with respect to bait protein is to be checked. The product of this vector is also a fusion product of both prey protein and transcription activation domain.

3.  The Fusion protein of bait vector binds to the specific promoter region of the reporter gene with the help of DNA binding domain but unable to initiate transcription as lacks activation domain.

4.  The prey protein fused with transcription activation domain, if interacts compatibly with bait protein then the activation factor is able to locate itself in the vicinity of reporter gene and able to initiate the transcription by allowing RNA polymerase to act.

5.  Thus, the change in color i.e. expression of the reporter gene clears that the bait protein is in interaction with prey protein and thus conclusion can be made about their functional activity further.

The Two hybrid system can be used to check the interaction of a single bait protein against thousands of prey proteins as only those cells would grow into which there is proper interaction taking place and allowing the selected gene essential for growth to be expressed.

The steps of two Hybrid System is summarized in animation