Use of the yeast two-hybrid technology to isolate molecular interactions of Ras GTPases

Since its original description, the yeast two-hybrid system has been extensively used to identify protein-protein interactions from many different organisms, thus providing a convenient mean to both screen for proteins that interact with a protein of interest and to characterize the known interaction between two proteins. In these years the technique has improved to overcome the limitations of the original assay, and many efforts have been made to scale up the technique and to adapt it to large-scale studies. In addition, variations have been introduced to enlarge the range of proteins and interactors that can be assayed by hybrid-based approaches. Several groups studying molecular mechanisms that underlie signal transduction pathways regulated by Ras GTPases have successfully used the yeast two-hybrid system or related methods to isolate and identify new binding partners of Ras proteins. Here we describe the basic protocol for a yeast two-hybrid library screening and for a small-scale yeast two-hybrid assay.