Advanced Yeast Two-Hybrid (Y2H) Screening Service

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For over 20 years, Creative Biolabs has been at the forefront of protein-protein interaction (PPI) analysis. Our Yeast Two-Hybrid (Y2H) service is a testament to this legacy, offering a powerful, versatile, and highly reliable platform to discover and validate novel PPIs. Harnessing the genetic power of Saccharomyces cerevisiae, our Y2H platforms are engineered to overcome the classic limitations of technology. By integrating next-generation sequencing (NGS), proprietary library construction techniques, and rigorous multi-step validation protocols, we deliver Y2H screening results with exceptionally high coverage and a remarkably low rate of false positives.

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The Principle of Yeast Two-Hybrid (Y2H) Screening

The Yeast Two-Hybrid system is an elegant and powerful in vivo technique for identifying protein-protein interactions. It ingeniously repurposes a yeast transcription factor, which typically consists of two distinct functional domains: a DNA-binding domain (BD) and a transcriptional activation domain (AD).

Separation of Domains: The system separates these two domains into two independent hybrid proteins.
Bait and Prey: The first protein, your "bait" (Protein X), is fused to the DNA-binding domain (BD). The second protein, the "prey" (Protein Y), sourced from a cDNA or ORF library, is fused to the activation domain (AD).
Interaction-Mediated Reconstruction: When the bait and prey proteins are co-expressed in a yeast cell, they are localized to the nucleus. If they interact, the BD and AD are brought into close proximity, functionally reconstituting the transcription factor.
Reporter Gene Activation: This reconstituted transcription factor then binds to an upstream activating sequence (UAS) in the yeast genome, driving the expression of one or more reporter genes (e.g., HIS3, ADE2, AUR1-C, lacZ). The growth of yeast on selective media or a color change indicates a positive interaction.

Fig. 1 Random Y2H library screening. (Creative Biolabs Original). Fig.1 A simple flowchart for a random Y2H library screening service.

Our Advanced Y2H Platforms: Tailored for Your Target

We recognize that no single platform fits every research question. We offer a suite of Y2H systems designed to meet diverse project needs.

High-Throughput NGS-Y2H Screening

Our flagship service. By replacing traditional colony picking and Sanger sequencing with deep sequencing of reporter gene barcodes, our NGS-Y2H platform redefines high-throughput screening.

Unprecedented Depth: Detect interactions from clones representing as little as 0.001% of the library population.
Quantitative Results: Obtain a ranked list of interactors based on sequence read counts, helping to prioritize the strongest and most significant hits.
Ideal For: Comprehensive interactome mapping, identifying transient or weak interactions, and comparative interactomics.

Membrane Yeast Two-Hybrid (MYTH) System

Standard Y2H fails for integral membrane proteins as they cannot be targeted to the nucleus. Our MYTH system overcomes this limitation by tethering the bait to the plasma membrane and adapting the reporter system accordingly.

Full-Length Protein Analysis: Screen integral membrane proteins in their native topological context without truncations.
Physiological Relevance: Detect interactions that occur at the cellular membrane.
Key Applications: Receptor-ligand discovery, ion channel interactomics, and signalosome mapping.

Classical Gal4/LexA-based Y2H

For standard, robust interaction validation and smaller-scale screens, our classical systems are meticulously optimized. They are perfect for:

One-on-One Interaction Studies: Confirming a predicted interaction between two specific proteins.
Small-Scale Library Screens: Focused projects to identify interactors for a well-characterized bait.
Domain Mapping: Identifying specific protein domains responsible for a known interaction.

A Comprehensive Portfolio of Y2H Services

We provide end-to-end support for your PPI research, from initial project design to downstream validation.

Bait & Library Services

Bait Plasmid Construction: Cloning your gene of interest into our optimized Y2H bait vectors.
Bait Auto-activation & Toxicity Testing: A critical quality control step to ensure a clean and reliable screen.
Custom cDNA/ORF Library Construction: We can generate high-quality, high-complexity libraries from any species or tissue type you provide.
Pre-made cDNA Libraries: Leverage our extensive collection of ready-to-screen libraries from human, mouse, rat, and other model organisms.

Screening & Validation Services

Y2H Library Screening: Comprehensive screening of your bait against a custom or pre-made library.
Matrix & Array-based Y2H: High-throughput screening of arrayed ORF libraries against your bait, ideal for systems biology approaches.
Hit Confirmation & Re-sequencing: All primary hits are isolated and re-tested to eliminate artifacts and confirm the interaction.
Orthogonal Validation: We strongly recommend validating key interactions with a secondary method. We offer a suite of services including Co-Immunoprecipitation (Co-IP), Pull-Down Assays, and Surface Plasmon Resonance (SPR).

Our Streamlined Y2H Service Workflow

We've developed a clear, phase-gated workflow to ensure transparency and project success from start to finish.

Consultation & Project Design: Our PhD-level scientists work with you to define project goals and select the optimal strategy.
Bait Preparation & QC: We construct the bait plasmid and perform rigorous auto-activation and expression tests.
Library Screening: The bait is screened against the chosen library using our high-stringency selection process.
Hit Identification & Sequencing: Positive colonies are isolated, and the prey plasmids are sequenced (Sanger or NGS) to identify the interacting proteins.
Data Analysis & Bioinformatics: Hits are filtered, annotated, and ranked. You receive a comprehensive bioinformatics report.
Final Report & Deliverables: A detailed final report, all relevant plasmid constructs, and raw data are delivered.

Why Choose Creative Biolabs for Your Y2H Project?

While Y2H is a standard technique, its successful execution demands deep expertise. We have refined every step of the process to provide a service that is not just a screen, but a complete discovery solution.

Feature	Creative Biolabs' Advantage	Client Benefit
High-Signal, Low-Noise System	Utilization of multiple, stringent reporter genes (up to four) and optimized yeast strains.	Drastically reduces false positives, saving you time and resources on validating irrelevant hits.
Advanced NGS-Y2H Platform	Integration of Next-Generation Sequencing with Y2H for unparalleled screening depth.	Identifies low-abundance interactors and provides quantitative data on interaction strength.
Expert Bait Optimization	Rigorous testing for auto-activation and toxicity before every screen.	Ensures your bait protein is viable for screening, preventing project delays and false negatives.
Superior Library Quality	Access to our extensive pre-made libraries (>10 M clones) or construction of high-complexity custom libraries.	Maximizes the probability of identifying all relevant biological interactors.
Specialized Membrane Y2H	Our proprietary MYTH (Membrane Yeast Two-Hybrid) system for full-length integral membrane proteins.	Unlocks the previously challenging area of membrane protein interactomics.
Comprehensive Data Analysis	Advanced bioinformatics pipelines for hit ranking, network visualization, and functional annotation.	Delivers not just a list of hits, but actionable biological insights.

Explore Our Related Protein Interaction Services

Leverage our full suite of interaction analysis platforms to build a complete picture of your biological system. Explore our full range of PPI services here: Protein-Protein Interaction (PPI) Assay Services

Ready to illuminate your protein interaction network? Contact us today to discuss your project with one of our Y2H specialists and receive a complimentary project design proposal.

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Frequently Asked Questions (FAQ)

Q: What is the main advantage of using a Yeast Two-Hybrid (Y2H) system?

A: The primary advantage of the Y2H system is its ability to perform in vivo discovery of novel protein-protein interactions directly within a eukaryotic cell. This physiological context is crucial for ensuring that interacting proteins are correctly folded and post-translationally modified. It is a powerful and highly scalable technique for screening millions of potential partners from a cDNA or ORF library, making it ideal for large-scale interactome mapping and hypothesis generation.

Q: Can the Y2H system be used for proteins that are not from yeast?

A: Absolutely. This is one of the great strengths of the system. You can use proteins from any organism—human, mouse, plant, virus, bacteria, etc. As long as the genes can be cloned into the Y2H vectors and the expressed proteins can fold properly and be targeted to the yeast nucleus, they can be studied using this system.

Q: My protein of interest is a membrane protein. Can I use the Y2H service?

A: Yes. For integral membrane proteins or proteins that are tightly associated with the membrane, the standard Y2H system is not suitable. For this exact reason, we have developed our specialized Membrane Yeast Two-Hybrid (MYTH) system. The MYTH system is specifically designed to detect interactions between full-length membrane proteins in their more native environment at the plasma membrane, overcoming a major limitation of the classical Y2H.

Reference

Yang, Fang, et al. "Development and application of a recombination-based library versus library high-throughput yeast two-hybrid (RLL-Y2H) screening system." Nucleic acids research 46.3 (2018): e17-e17. https://doi.org/10.1093/nar/gkx1173

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