Yeast Two-Hybrid Screening to Test for Protein-Protein Interactions in the Auditory System

We describe a protocol to screen for protein-protein interactions using the Gal-4-based yeast two-hybrid system. In this protocol, we describe serial transformation of bait into an already constructed cDNA library in yeast AH109 cells. We find this method gives the most number of true interactions. Where a premade library in yeast cells is not available, the method outlined can be quickly adapted. AH109 cells can be first transformed with bait containing a vector followed by selection of yeast containing the bait. A second transformation of yeast cells is then accomplished with the cDNA library. The method is quick and can lead to the discovery of significant interactions.