Yeast 2-hybrid assay for investigating the interaction between the centrosome proteins PLK4 and STIL

Centrosomes act as a major microtubule-organizing center in many animal cells. This non-membranous cell organelle is involved in a wide range of cell functions like division, migration, and signaling. Centrosome houses hundreds of proteins that interact in a regulated manner to carry out their tasks. Most of these centrosome proteins have either the coiled-coil or disordered regions, making them challenging to purify for functional analysis. Yeast 2-hybrid (Y2H) is an in vivo based method for studying protein–protein interactions by exploiting the modular nature of yeast's GAL4 transcription factor. This method provides an alternative strategy for investigating bi-molecular interactions, especially when dealing with otherwise difficult proteins to purify by conventional biochemical approaches. We present a detailed protocol for showing the interaction between the two core centrosome proteins, i.e., PLK4 and STIL, using the Y2H assay.