YeastTwoHybridAssays

The Yeast Two-Hybrid (Y2H) assay is a molecular biology technique used to detect protein-protein interactions in vivo. It is based on the principle of reconstituting a functional transcription factor from two separate protein domains, each fused to a different bait protein. One domain is the DNA-binding domain (DBD), and the other is the activation domain (AD). When two proteins of interest, one fused to the DBD and the other to the AD, interact, they bring these two domains into close proximity. This reconstitution allows the transcription factor to bind to a specific DNA sequence upstream of a reporter gene, leading to its expression. Reporter genes commonly used include lacZ, which produces a blue color in the presence of a substrate, or HIS3, which allows yeast cells to grow in the absence of histidine. The Y2H system can be used to screen libraries of potential interacting proteins, identifying novel partners for a bait protein. It is a powerful tool for studying the complex network of interactions within a cell. Variations of the Y2H assay exist, such as the bacterial two-hybrid system, which overcomes some limitations of the yeast system. Careful experimental design and controls are essential to interpret Y2H results accurately, as false positives and negatives can occur.