Yeast Three-hybrid (Nuclear System) Screening

Proteins rarely act alone because their function is often regulated in vivo. Thus, protein-protein interaction analysis provides key clues for understanding complex biological processes in living cells. The yeast two-hybrid (Y2H) system is a well-established method for analyzing binary protein–protein interactions. Y2H is relatively simple and easy to establish and is therefore a suitable first choice for screening the large number of possible interacting partners for given proteins. Yeast three-hybrid (Y3H) technology is an extension of the Y2H for detecting three-molecular interactions involving proteins.

Amerigo Scientific offers Y3H screening service for studying protein-protein-small molecule binding, protein-DNA or protein-RNA interactions, and other complex biological processes. Based on advanced screening and validation workflows, our Y3H service supports breakthrough in drug development, functional genomics, and signaling pathway analysis.

Figure 1. Technique Principle of Our Y3H Service

Our Y3H system utilizes an innovative pBridge plasmid, which is characterized by its ability to simultaneously incorporate two exogenous proteins. The protein inserted into the MCSI site remains fused at its N-terminus with the binding domain (BD) of the Gal4 protein, whereas the protein inserted into the MCSII site is not fused with any tag. However, upstream of this insertion site, there is a Pmet25 promoter, which is only active in the absence of methionine (Met), allowing the expression of the downstream protein-coding gene. The pBridge plasmid is transformed into the Y2HGold yeast strain, while a third protein-coding gene is cloned into the pGADT7 vector and transformed into the AH109 yeast strain. These two yeast strains are then subjected to a small-scale mating process. The remaining experimental procedures are consistent with the Y2H system, with the only difference being that all selective media must be formulated based on methionine-deficient conditions. If an interaction occurs among the three proteins, yeast colonies will be able to grow on SD/-Met/-Ade/-His/-Leu/-Trp selective media.

Applications of Our Y3H Service

• Drug discovery, such as identifying modulators that affect protein-protein or protein-small molecule interactions.
• Functional genomics, such as studying the mechanisms of complex nucleic acid-binding proteins.
• Signaling pathway studies, such as the analysis of complex signaling cascades involving multiple interacting partners.

Key Advantages of Our Y3H Service

• Wide Range of Applications: Our service supports the studies of protein complexes, protein post-translational modifications, or nucleic acid binding.
• High Precision: The use of advanced pBridge plasmid ensures in vivo validation under methionine-deficient conditions.
• Efficient Screening: Detailed results from high-throughput experiments accelerate your research efforts.
• Reliable Results: Robust protocols, validated positive clones, and in-depth analysis reports are provided.
• Streamlined Processes: Optimized protocols minimize turnaround time while maximizing accuracy.
• Customizable Services: Custom library options are available, and screening conditions can be optimized for specific needs.

Content of Our Y3H Service

The total workflow of our service typically requires about 60 working days.