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 to gives the most number of true interactions. Where a pre-made 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.
KeywordsProtein-protein interactions Yeast two-hybrid Gal4 based cochlea cDNA library pGBKT7 pGADT7 AH109 serial transformation
- 3.Bartel, P. L., Chien, C.-T., Sternglanz, R., and Fields, S. (1993) Using the two-hybrid system to detect protein-protein interactions, in Cellular Interactions in Development: A Practical Approach (Hartley, D. A., ed.) Oxford University Press, Oxford pp. 153–179.Google Scholar
- 7.Golemis, E. A., Gyuris, J., and Brent, R. (1994) Interaction trap/two-hybrid systems to identify interacting proteins, in Current Protocols in Molecular Biology, John Wiley & Sons, Inc., Hoboken, NJ, Chapters 13 and 14.Google Scholar
- 8.Golemis, E. A., Gyuris, J., and Brent, R. (1996) Analysis of protein interactions; and Interaction trap/two-hybrid systems to identify interacting proteins, in Current Protocols in Molecular Biology, John Wiley & Sons, Inc., Hoboken, NJ, Chapters 20.0 and 20.1.Google Scholar