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Protein–protein interactions (PPIs) underpin cellular signaling, gene regulation, and most biological processes. Mapping these interactions is essential for understanding protein function, dissecting signaling pathways, and identifying potential therapeutic targets. The yeast two-hybrid (Y2H) system remains one of the most widely used in vivo screening methods for detecting binary PPIs, owing to its sensitivity, scalability, and ability to recapitulate interactions within a eukaryotic cellular environment.
The Y2H system is based on the modular architecture of eukaryotic transcription factors, such as GAL4, which contain physically separable DNA-binding domains (BD) and activation domains (AD). When these two domains are brought into proximity by interacting proteins, they reconstitute a functional transcription factor that activates downstream reporter genes in yeast. This principle allows the detection of PPIs directly inside living cells without requiring recombinant protein purification.
Figure 1. Yeast two-hybrid principle. (Rajagopala SV. et al., 2015)
Creative Biogene offers a comprehensive Yeast Two-Hybrid cDNA Library Construction and Screening Service, supporting both traditional nuclear Y2H systems and membrane-based split-ubiquitin systems. With extensive experience in RNA preparation, cDNA synthesis, vector engineering, yeast transformation, and interaction validation, we provide high-quality Y2H libraries and screening results tailored to diverse research needs. Our platform integrates optimized cloning strategies, stringent quality control, and mechanistic understanding of transcriptional activation, ensuring reliable identification of biologically meaningful protein interactions.
A high-quality cDNA library is essential for reliable interaction screening. Creative Biogene constructs libraries with high diversity, accurate representation, and strong insert quality through an optimized workflow.
Our cDNA libraries are suitable for discovering unknown interaction partners, studying regulatory networks, or defining protein domains involved in target binding. Directional cloning strategies minimize frame shifts, and our systems can support three-frame library construction when needed to ensure complete representation of coding sequences.
Creative Biogene offers end-to-end screening services using constructed or client-provided libraries. Screens can be performed using nuclear Y2H or membrane Y2H, depending on the localization and biochemical characteristics of the bait protein.
Nuclear Y2H Screening
Bait proteins are fused to the GAL4 DNA-binding domain, while prey cDNAs are expressed as GAL4-AD fusions. Interactions reconstitute the transcription factor and activate reporter genes such as HIS3, ADE2, or MEL1. Multi-step selection on dropout media, followed by enzymatic reporter assays, ensures reliable identification of interacting clones.
Membrane Y2H Screening
For membrane-bound or structurally complex proteins, the split-ubiquitin system enables detection of interactions in the cytoplasmic environment. Nub/Cub recognition and ubiquitin-processing enzyme cleavage trigger reporter activation, allowing screening under conditions more reflective of native membrane biology.
Y2H screening requires careful control of self-activation, toxicity, and growth conditions. Creative Biogene implements the following measures in all projects:
These quality control steps ensure that identified interacting proteins faithfully reflect biologically relevant interactions.
Yeast two-hybrid library construction and screening are widely applied in:
The method is particularly valuable in fields such as cancer biology, immunology, plant signaling, neurobiology, host–pathogen interactions, and drug target discovery.
Although each project is customized, a typical workflow includes:
1Project Consultation: Selection of bait format, system type (nuclear or membrane), vector design, and screening strategy
2Library Construction or QC: cDNA preparation, vector cloning, transformation, diversity assessment
3Yeast Transformation & Pre-Screening: Bait testing, self-activation detection
4Interaction Screening: Growth-based selection and reporter activation
5Clone Identification: Isolation, plasmid recovery, insert sequencing
6Verification & Analysis: Retesting candidate interactions, bioinformatics annotation
7Reporting: Comprehensive results including screening images, clone sequences, and analysis
Creative Biogene supports both full-process projects and modular service options.
Depending on the project type, deliverables may include:
Creative Biogene is committed to providing reliable, scientifically rigorous Y2H services that support reproducible research outcomes. Our advantages include:
Whether your goal is identifying new interaction partners, validating candidate proteins, or mapping complex signaling networks, Creative Biogene provides a robust and efficient Y2H platform suited to diverse research needs.
Contact us to discuss your experimental objectives — our scientific team will assist you in designing a customized solution for your protein interaction studies.
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