The traditional Y2H detection principle utilizes a reporter gene whose activation depends on a specific transcription factor. Specifically, the protein X and Y to be tested are fused with the DNA-binding domain (BD) and the activation domain (AD) of the transcription factor. The BD-X fusion protein binds to the upstream activation sequence (UAS) of the reporter gene promoter. When X and Y interact, the AD is recruited to the promoter, reconstructing a functional transcription factor, which triggers the subsequent transcription of the reporter gene. (Figure 1)

As a genetic technology, yeast two-hybrid screening offers a sensitive and economical method to test direct interactions between two target proteins, or to use a protein as bait to screen protein fragment libraries derived from the desired cell types, tissues, or entire organisms. The corresponding plasmids from the selected yeast colonies are sequenced to determine the interactions.

Figure 1: Schematic Diagram of Basic Y2H Principle

1. Create BD-X bait.

2. Test the self-activation of BD-X bait.

3. Perform yeast two-hybrid screening and identify the prey.

To study the interactions of a large number of proteins, scientists have developed several systems based on the Y2H system to adapt to the different subcellular localizations and biochemical characteristics of proteins.We offer a variety of systems to meet the diverse needs of different projects.

·Yeast Single-Hybrid System: Studies protein-DNA interactions (see Figure 2).

Figure 2: Schematic Diagram of Y1H Principle

·Yeast Triple-Hybrid System: Studies protein-protein interactions mediated by a third component (see Figure 3).

Figure 3: Schematic Diagram of Y3H Principle

·Split-Ubiquitin Y2H System: Used to study membrane proteins (see Figure 4).

We provide a one-stop service: from library construction to positive clone screening. This enhances efficiency, avoids errors, and objectively saves costs for our clients.

Service Content:

-- Clone the bait target gene into a Y2H vector and sequence it. The bait target gene can be a gene fragment (cut by restriction enzymes) or a full-length gene.

-- Test the self-activation ability and toxicity of the bait gene.

-- Library screening and interaction verification.

-- Isolation, extraction of positive clones, and sequencing of DNA.

-- At least 2μg of plasmid (including bait target gene).

-- The DNA sequence of the gene for us to design the synthesis plan and perform gene synthesis (subject to additional charges).

Deliverables:

-- Several positive clones and DNA sequencing results.

-- At least 2μg of plasmid for each positive clone.

-- Experimental Timeline: Approximately 6-8 weeks.