Yeast Two-Hybrid (Y2H) Assay: Principles and Applications

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to Yeast Two-Hybrid (Y2H) Assay

What is Y2H?

The yeast two-hybrid (Y2H) assay is a molecular genetic technique used to detect and characterize physical interactions between two proteins inside living yeast cells. Developed by Stanley Fields and Ok-Kyu Song in 1989, the method exploits the modular architecture of eukaryotic transcription factors to convert a protein-protein interaction into a quantifiable transcriptional readout. In essence, Y2H asks a simple question: do protein X and protein Y bind to each other? — and answers it by linking that binding event to the activation of reporter genes whose products are easily assayed.

The assay is performed in Saccharomyces cerevisiae, a eukaryotic organism that provides a native environment for protein folding, post-translational processing, and nuclear localization. Because the readout is genetic (growth or color), Y2H is exceptionally sensitive and can detect interactions with dissociation constants (Kd) in the low micromolar to nanomolar range. This sensitivity, combined with the ease of manipulating yeast genetics, has made Y2H a cornerstone of interactomics — the large-scale study of protein-protein interaction networks.

Why study protein interactions?

Proteins rarely act in isolation. Cellular processes — signal transduction, transcriptional regulation, DNA replication, vesicular trafficking, and apoptosis — depend on precise, often transient, physical associations between proteins. Mapping these interactions is essential for understanding:

• Biological function: Knowing what a protein binds to often reveals what it does. An uncharacterized protein that interacts with a known kinase, for example, is likely involved in the same signaling pathway.
• Disease mechanisms: Many genetic diseases result from mutations that disrupt or create aberrant protein interactions. Identifying these altered contacts can explain pathophysiology and suggest therapeutic targets.
• Network biology: Large-scale Y2H screens have produced interaction maps for organisms from yeast to humans, providing a framework for systems-level analysis.

The Yeast Two Hybrid System remains one of the most widely used approaches for these purposes because it is inexpensive, scalable, and does not require specialized equipment beyond standard microbiology supplies.

The Biological Basis of Y2H

Transcription factor domains

The Y2H assay rests on a fundamental observation about eukaryotic transcription factors: they are modular proteins composed of at least two functionally independent domains. The DNA-binding domain (BD) recognizes and binds to a specific DNA sequence (the upstream activating sequence, or UAS) in the promoter of target genes. The activation domain (AD) recruits the transcriptional machinery — including RNA polymerase II and general transcription factors — to initiate transcription.

For a transcription factor to activate gene expression, both domains must be present and physically associated. Critically, they do not need to be part of the same polypeptide chain. If the BD and AD are expressed as separate fusion proteins, they cannot activate transcription unless they are brought into close proximity by some other means. This property is the foundation of Y2H.

The most commonly used transcription factor in Y2H is Gal4 from yeast, which regulates genes involved in galactose metabolism. Gal4's BD (amino acids 1–147) binds to the GAL UAS, and its AD (amino acids 768–881) activates transcription. Other systems use the bacterial repressor LexA (BD) fused to the herpes simplex virus VP16 AD, but the Gal4 system is the most widespread.

Reconstitution of a functional activator

The Y2H strategy is conceptually elegant. You create two fusion proteins:

1. Bait: Your protein of interest (protein X) fused to the Gal4 DNA-binding domain (Gal4-BD). This construct is expressed from a plasmid and localizes to the nucleus, where it binds the UAS upstream of reporter genes.
2. Prey: A candidate interacting protein (protein Y) fused to the Gal4 activation domain (Gal4-AD). This fusion protein cannot bind DNA on its own.

When the bait and prey are co-expressed in the same yeast cell, nothing happens unless protein X and protein Y physically interact. If they do, the interaction brings the Gal4-AD into proximity with the Gal4-BD, reconstituting a functional transcription factor. The AD can then recruit the transcriptional machinery to the UAS, activating transcription of downstream reporter genes.

This principle is sometimes described as "two halves of a transcription factor reassembled by protein-protein interaction." The readout is binary in principle — transcription is either activated or not — but can be made quantitative by measuring the activity of reporter gene products. The Yeast Two Hybrid Assay thus provides both a qualitative and quantitative measure of interaction strength.

Key Components and Strains Used in Y2H

Bait and prey constructs

The bait and prey proteins are expressed from separate plasmids, each carrying a different selectable marker to allow maintenance in yeast. Standard vectors include:

• pGBKT7 (bait): Carries the TRP1 gene for selection on medium lacking tryptophan; expresses the bait as a fusion to Gal4-BD, with a c-Myc epitope tag for detection by Western blot.
• pGADT7 (prey): Carries the LEU2 gene for selection on medium lacking leucine; expresses the prey as a fusion to Gal4-AD, with a hemagglutinin (HA) epitope tag.

Both plasmids contain a multiple cloning site downstream of the fusion domain, a yeast origin of replication (2µ or ARS/CEN), and an E. coli origin and antibiotic resistance gene (e.g., ampicillin) for propagation in bacteria. The choice of vector depends on the application: high-copy 2µ plasmids give higher expression levels, while centromeric (CEN) plasmids give more physiologically relevant, lower expression.

The open reading frame of your protein of interest is cloned in-frame with the Gal4 domain. This is typically done by PCR amplification of the coding sequence, digestion with restriction enzymes, and ligation into the vector. Alternatively, Gateway recombination cloning can be used for high-throughput applications.

Reporter genes

Reporter genes are the output of the Y2H assay. They are placed under the control of a promoter containing the Gal4 UAS, so their expression depends on the reconstitution of a functional Gal4 activator. Standard Y2H strains contain multiple reporter genes, each with a different readout:

The HIS3 and ADE2 reporters allow selection: only cells with an interaction can synthesize histidine or adenine and thus grow on medium lacking these nutrients. The lacZ and MEL1 reporters allow visual or quantitative detection: β-galactosidase or α-galactosidase activity produces a blue color when cells are grown on medium containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) or X-α-gal, respectively.

Using multiple reporters with different promoters reduces false positives, because a true interaction must activate all reporters simultaneously. The HIS3 reporter is often used with 3-aminotriazole (3-AT), a competitive inhibitor of the HIS3 gene product, to titrate sensitivity and suppress weak background growth.

Common yeast strains

Several yeast strains have been engineered specifically for Y2H. The most widely used are:

• AH109: Carries lacZ, HIS3, and ADE2 reporters under three different Gal4-responsive promoters (GAL1, GAL2, and GAL7, respectively). The use of different promoters minimizes promoter-specific artifacts.
• Y190: Carries lacZ and HIS3 reporters; commonly used for library screening.
• Y2HGold: A modern strain with lacZ, HIS3, ADE2, and MEL1 reporters, plus mutations that improve transformation efficiency and reduce background.

These strains are auxotrophic — they carry mutations in biosynthetic genes (trp1, leu2, his3, ade2) that prevent growth on medium lacking the corresponding nutrient. This auxotrophy is exploited for plasmid selection: a strain transformed with the bait plasmid (carrying TRP1) can grow on medium lacking tryptophan, while a strain transformed with the prey plasmid (carrying LEU2) can grow on medium lacking leucine. The Yeast Model Organism is ideal for this purpose because its genetics are well understood and its growth requirements are easily manipulated.

Step-by-Step Workflow of a Y2H Screen

Constructing bait and prey

The first step is to clone your gene of interest into the bait vector (pGBKT7) and your candidate interacting protein(s) into the prey vector (pGADT7). For a targeted experiment, you might clone a single known protein as prey. For a library screen, you would use a cDNA library constructed in the prey vector, representing thousands of different proteins expressed from the tissue or organism of interest.

Key considerations during cloning:

• Ensure the open reading frame is in-frame with the Gal4 domain. A frame-shift will produce a truncated or mis-sense fusion protein.
• Remove the stop codon from the gene of interest if it is to be fused at the C-terminus of the Gal4 domain; keep it if fusing at the N-terminus.
• Verify the construct by DNA sequencing before proceeding.

Transformation and mating

There are two strategies for introducing bait and prey plasmids into yeast:

1. Co-transformation: Both plasmids are transformed simultaneously into the same yeast strain. This is simple but less efficient, making it suitable for testing a few defined pairs.
2. Mating: Bait is transformed into one haploid strain of mating type a (e.g., Y187), and prey into another haploid strain of mating type α (e.g., AH109). The two strains are then mixed and allowed to mate, producing diploid cells that contain both plasmids. Mating is more efficient for library screens because the bait strain can be mated against an entire prey library in a single experiment.

Transformation of yeast is typically done using the lithium acetate/single-stranded carrier DNA/PEG method. Briefly:

1. Grow yeast to mid-log phase (OD₆₀₀ ≈ 0.6–0.8) in rich medium (YPD).
2. Harvest cells by centrifugation and wash with sterile water.
3. Resuspend cells in a solution containing 100 mM lithium acetate, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 40% polyethylene glycol (PEG 3350).
4. Add 0.1–1 µg plasmid DNA and 100 µg boiled salmon sperm DNA (as carrier).
5. Incubate at 42°C for 15–30 minutes (heat shock).
6. Plate on selective medium (e.g., SD/-Trp for bait, SD/-Leu for prey).

Selecting for positive interactions

After co-transformation or mating, cells are plated on double dropout medium lacking tryptophan and leucine (SD/-Trp/-Leu) to select for diploids containing both plasmids. These cells are then replica-plated onto triple dropout medium (SD/-Trp/-Leu/-His) or quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade) to select for cells in which the interaction has activated the HIS3 and ADE2 reporters.

Only cells expressing two interacting proteins will grow on these stringent media. The stringency can be adjusted by adding 3-AT (typically 1–50 mM) to the medium to inhibit low-level HIS3 background activity. Colonies typically appear within 2–5 days at 30°C.

Confirming with controls

Positive colonies must be verified with appropriate controls to rule out artifacts:

• Empty vector control: Transform bait with empty prey vector and prey with empty bait vector. No growth should occur on selective medium.
• Known interaction pair: Use a well-characterized interaction (e.g., p53 with SV40 large T antigen) as a positive control to confirm the system is working.
• Replica plating: Streak positive colonies onto fresh selective plates to confirm the phenotype is stable.
• Plasmid rescue and retesting: Isolate the prey plasmid from yeast, amplify it in E. coli, sequence it, and retransform into fresh bait-containing yeast to confirm the interaction is reproducible.

The Yeast Two-hybrid Screening workflow is summarized in the following numbered sequence:

1. Clone bait and prey into Y2H vectors.
2. Transform bait into a mating-type a strain; transform prey (or library) into a mating-type α strain.
3. Mate the two strains on rich medium (YPD) for 4–6 hours at 30°C.
4. Plate diploids on SD/-Trp/-Leu to select for both plasmids.
5. Replica-plate onto SD/-Trp/-Leu/-His (+3-AT) and SD/-Trp/-Leu/-His/-Ade.
6. Incubate 3–5 days at 30°C; pick colonies.
7. Assay for β-galactosidase or α-galactosidase activity.
8. Isolate prey plasmids, sequence, and confirm interactions in fresh yeast.

Interpreting Y2H Results

Growth on selective media

The most direct readout of Y2H is growth on selective medium. A strong interaction will produce large colonies on SD/-Trp/-Leu/-His/-Ade within 2–3 days. A weaker interaction may only support growth on SD/-Trp/-Leu/-His (with or without 3-AT) or produce smaller colonies. The ADE2 reporter is particularly useful because it provides a visual gradient: cells with strong ADE2 expression are white, while cells with weak or no expression accumulate a red intermediate in the adenine biosynthesis pathway and appear pink or red.

Growth phenotypes should be compared to controls:

• No interaction: No growth on selective medium.
• Weak interaction: Growth on SD/-Trp/-Leu/-His but not on SD/-Trp/-Leu/-His/-Ade.
• Strong interaction: Growth on both SD/-Trp/-Leu/-His and SD/-Trp/-Leu/-His/-Ade.

Quantitative assays

For a more quantitative measure of interaction strength, the lacZ reporter is assayed. β-galactosidase activity is proportional to the amount of transcription activated by the interaction. The standard assay uses the substrate ONPG (o-nitrophenyl-β-D-galactoside), which is cleaved by β-galactosidase to produce a yellow product (o-nitrophenol) that absorbs at 420 nm.

The assay protocol:

1. Grow yeast cells in liquid selective medium to mid-log phase.
2. Harvest and permeabilize cells with chloroform and SDS (or use glass beads for lysis).
3. Add ONPG (final concentration 0.8 mg/mL) in Z-buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, pH 7.0, with 50 mM β-mercaptoethanol).
4. Incubate at 30°C until a yellow color develops (typically 5–60 minutes).
5. Stop the reaction with 1 M Na₂CO₃ and measure absorbance at 420 nm and 600 nm (for cell density).

β-galactosidase activity is calculated in Miller units:

Miller units = (1000 × A₄₂₀) / (t × V × A₆₀₀)

where t is the reaction time in minutes and V is the volume of culture used in mL.

Higher Miller units indicate stronger interaction. Typical values range from <1 (no interaction) to several hundred (strong interaction). However, absolute values vary between laboratories and should always be interpreted relative to positive and negative controls run in the same experiment.

False positives and negatives

Y2H is prone to both false positives (interactions detected that do not occur in vivo) and false negatives (real interactions missed). Common causes include:

False positives:

• Autoactivation: The bait alone activates transcription without a prey, often due to an intrinsic activation domain in the bait protein.
• Sticky or promiscuous proteins: Highly charged or hydrophobic proteins may interact nonspecifically with many partners.
• Overexpression artifacts: High expression levels from 2µ plasmids force interactions that would not occur at physiological concentrations.
• Bridging by yeast proteins: Endogenous yeast proteins may bridge the bait and prey, creating an apparent interaction.

False negatives:

• Poor expression or instability of fusion proteins in yeast.
• Improper folding of the fusion protein, especially if the Gal4 domain interferes with the native structure.
• Interactions requiring post-translational modifications not present in yeast (e.g., specific phosphorylation by mammalian kinases).
• Membrane or secreted proteins that do not fold correctly or localize to the nucleus.

Applications of Y2H in Research

Interaction mapping

Y2H has been used to generate comprehensive protein-protein interaction maps for model organisms. The most famous example is the yeast interactome, where thousands of interactions were mapped by high-throughput Y2H screens. These maps provide a framework for understanding cellular pathways and predicting the function of uncharacterized proteins based on their interaction partners.

For example, a protein that interacts with components of the proteasome is likely involved in protein degradation; a protein that interacts with transcription factors is likely involved in gene regulation. Interaction maps have been generated for S. cerevisiae, C. elegans, Drosophila, and human proteins, among others.

Screening libraries

One of the most powerful applications of Y2H is the library screen — identifying novel binding partners for a protein of interest. A cDNA library (representing all mRNAs expressed in a tissue or cell type) is constructed in the prey vector and introduced into yeast by mating. The bait strain is mated against the library, and diploids are plated on selective medium. Only cells where the bait interacts with a library-encoded protein will grow.

This approach has identified:

• Novel substrates of kinases and ubiquitin ligases.
• Receptors for secreted signaling molecules.
• Binding partners for viral proteins, revealing host factors exploited during infection.
• Interactors of disease-associated proteins, such as huntingtin (the protein mutated in Huntington's disease) and p53.

A typical library screen involves 10⁶–10⁷ transformants, and positive colonies are recovered at a frequency of 1 in 10⁴–10⁵. Each positive must be verified by retesting and sequencing.

Studying mutant proteins

Y2H is invaluable for studying how mutations affect protein interactions. By cloning wild-type and mutant versions of a protein as bait, you can compare their ability to interact with a known prey. This approach has been used to:

• Map interaction interfaces by alanine-scanning mutagenesis.
• Identify disease-causing mutations that disrupt (or create) interactions.
• Determine the functional significance of post-translational modification sites by mutating them to mimic or block modification.

For example, mutations in the tumor suppressor p53 that abolish its interaction with the E3 ubiquitin ligase MDM2 are found in human cancers. Y2H can be used to test whether a particular p53 variant retains MDM2 binding, providing insight into its oncogenic potential.

The Yeast 3 Hybrid System extends this approach to detect interactions involving three components, such as RNA-protein interactions bridged by a third molecule.

Advantages and Limitations of Y2H

Advantages

• In vivo context: Interactions are detected inside living eukaryotic cells, allowing proper protein folding and, in some cases, post-translational modifications.
• High sensitivity: Can detect weak and transient interactions (Kd in the micromolar range) that might be missed by biochemical methods.
• Scalability: Suitable for high-throughput screening of thousands of proteins simultaneously.
• Cost-effective: Requires only standard microbiology supplies; no specialized equipment such as mass spectrometers or biosensors.
• Direct genetic readout: The interaction is linked to a selectable phenotype (growth), enabling the isolation of interacting clones from complex libraries.
• Versatility: Can be adapted to detect RNA-protein interactions (yeast three-hybrid), small molecule-protein interactions, and protein-DNA interactions.

Limitations

• Nuclear localization requirement: Both fusion proteins must enter the nucleus. Membrane proteins, secreted proteins, and proteins with strong membrane-targeting signals often fail in Y2H.
• False positives: The assay can detect interactions that do not occur in the native cellular context due to overexpression, mislocalization, or the absence of competing partners.
• False negatives: Interactions requiring specific post-translational modifications (e.g., tyrosine phosphorylation by mammalian kinases) or cofactors absent in yeast will be missed.
• Toxicity: Some fusion proteins are toxic to yeast, preventing growth regardless of interaction.
• Autoactivation: Bait proteins with intrinsic transcriptional activation activity produce background signal.
• No quantitative affinity measurement: While β-galactosidase assays provide a rough measure of interaction strength, Y2H does not give precise binding constants.

Common Pitfalls and How to Avoid Them

Autoactivation

Problem: The bait protein alone activates reporter gene transcription, producing false positives even without a prey.

Detection: Transform bait alone into the reporter strain and plate on selective medium. If colonies grow, the bait is autoactivating.

Solutions:

• Use a less sensitive reporter strain (e.g., omit the ADE2 reporter).
• Add 3-AT to the medium to suppress weak HIS3 activation.
• Use a truncated bait lacking the activating region.
• Switch to a different BD (e.g., LexA instead of Gal4).
• If autoactivation persists, the protein may not be suitable for Y2H; consider alternative methods.

Toxicity of bait

Problem: Some proteins, when overexpressed in yeast, inhibit growth or kill the cells, preventing any Y2H analysis.

Detection: Transform bait into yeast and compare growth on selective medium to a vector-only control. If the bait strain grows poorly or not at all, the protein is toxic.

Solutions:

• Use a low-copy (CEN) vector to reduce expression.
• Use an inducible promoter (e.g., GAL1) to control expression timing.
• Clone a smaller domain of the protein that retains interaction capability.
• Use a yeast strain with reduced sensitivity to the toxic protein.

Weak interactions

Problem: Weak or transient interactions may not produce detectable reporter activation.

Solutions:

• Increase sensitivity by using the ADE2 reporter or reducing 3-AT concentration.
• Increase bait and prey expression by using high-copy vectors.
• Optimize growth conditions (temperature, medium).
• Use a more sensitive β-galactosidase substrate (e.g., chemiluminescent substrates like Galacton-Star).

Confirming interactions

Problem: Y2H results must be verified because of the high rate of false positives.

Solutions:

• Co-immunoprecipitation (co-IP): Express both proteins with epitope tags in mammalian or insect cells, immunoprecipitate one, and blot for the other.
• Pull-down assays: Incubate recombinant GST-tagged bait with prey protein and detect binding by Western blot.
• Fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET): Detect interactions in living mammalian cells.
• Co-localization by immunofluorescence: Show that both proteins localize to the same cellular compartment.
• Genetic complementation: Use a biological assay where the interaction is required for a phenotype.

The Yeast Two-hybridization approach is powerful, but orthogonal validation is essential before drawing biological conclusions.

Summary and Key Takeaways

The yeast two-hybrid assay is a powerful, versatile method for detecting protein-protein interactions in a eukaryotic context. By exploiting the modular nature of the Gal4 transcription factor, Y2H converts a physical interaction between two proteins into a genetic readout that can be selected, counted, and quantified. The assay has been instrumental in mapping interaction networks, identifying novel binding partners, and dissecting the molecular basis of disease.

Key points to remember:

• Y2H detects interactions between a bait (fused to a DNA-binding domain) and a prey (fused to an activation domain) by reconstituting a functional transcription factor.
• Multiple reporter genes (HIS3, ADE2, lacZ, MEL1) provide both selection and quantitative readouts.
• The assay is performed in S. cerevisiae, which provides a eukaryotic environment for protein folding.
• Controls are essential to distinguish true positives from autoactivation and other artifacts.
• Y2H has limitations, particularly for membrane proteins and interactions requiring specific post-translational modifications.
• All Y2H results should be confirmed by an independent method such as co-immunoprecipitation.

Frequently Asked Questions

What is the yeast two-hybrid (Y2H) assay?

The yeast two-hybrid assay is a molecular biology technique that detects physical interactions between two proteins by expressing them as fusions to the DNA-binding and activation domains of a transcription factor in yeast. If the proteins interact, the transcription factor is functionally reconstituted, activating reporter genes that produce a detectable signal such as growth on selective medium or enzyme activity.

How does the yeast two-hybrid system work?

The system works by splitting a transcription factor (usually Gal4) into two domains: a DNA-binding domain (BD) and an activation domain (AD). One protein of interest (bait) is fused to the BD, and another (prey) is fused to the AD. When the bait and prey interact, the BD and AD are brought together, reconstituting a functional transcription factor that activates reporter genes downstream of the Gal4 upstream activating sequence.

What are the main components of a Y2H assay?

The main components are: (1) a bait plasmid expressing the protein of interest fused to a DNA-binding domain, (2) a prey plasmid expressing a candidate interactor fused to an activation domain, (3) a yeast strain containing reporter genes under the control of a Gal4-responsive promoter, and (4) selective growth media to detect reporter activation.

What are common causes of false positives in Y2H?

Common causes include autoactivation (the bait activates transcription on its own), overexpression artifacts (high protein levels force nonspecific interactions), sticky or promiscuous proteins that bind many partners, and bridging by endogenous yeast proteins. The Yeast Two Hybrid System requires careful controls to minimize these issues.

How do you confirm a Y2H interaction?

Confirm interactions by co-immunoprecipitation, pull-down assays, FRET/BRET, co-localization by microscopy, or genetic complementation. Always retest the interaction in fresh yeast with empty vector controls to rule out plasmid contamination or artifacts.

Can Y2H detect interactions with membrane proteins?

Generally, no. Membrane proteins are hydrophobic and often fail to fold correctly or localize to the nucleus, where the Y2H interaction must occur. Variants such as the split-ubiquitin system or the membrane yeast two-hybrid (MYTH) assay have been developed for membrane proteins.

What is autoactivation in Y2H?

Autoactivation occurs when the bait protein alone activates reporter gene transcription without interacting with a prey. This happens when the bait contains an intrinsic transcriptional activation domain or is highly acidic. Autoactivation produces false positives and must be tested for by transforming the bait alone into the reporter strain.

What are the limitations of Y2H?

Limitations include the requirement for nuclear localization, inability to detect interactions requiring specific post-translational modifications absent in yeast, false positives and negatives, toxicity of some fusion proteins, and the lack of quantitative binding affinity measurements. The Diagram of Yeast Cell illustrates why compartmentalization matters: the nucleus is the only site where the BD and AD can meet.

Key Takeaways

• The yeast two-hybrid assay detects protein-protein interactions by reconstituting a split transcription factor in living yeast cells.
• The bait protein is fused to a DNA-binding domain; the prey protein is fused to an activation domain; interaction brings the two domains together to activate reporter genes.
• Multiple reporters (HIS3, ADE2, lacZ, MEL1) provide both qualitative (growth, color) and quantitative (β-galactosidase activity) readouts.
• Y2H is highly sensitive and scalable, making it ideal for both targeted studies and high-throughput library screens.
• The assay has significant limitations, including false positives from autoactivation and false negatives for membrane proteins or interactions requiring mammalian-specific modifications.
• All Y2H findings must be validated by orthogonal methods such as co-immunoprecipitation or FRET.
• Understanding the biological basis, workflow, and pitfalls of Y2H is essential for designing rigorous experiments and interpreting results correctly.

Further Reading

• Koegl M, Uetz P. Improving yeast two-hybrid screening systems. Briefings in functional genomics & proteomics. 2007. PubMed 18218650
• Brückner A et al. Yeast two-hybrid, a powerful tool for systems biology. International journal of molecular sciences. 2009. PubMed 19582228
• Hamdi A, Colas P. Yeast two-hybrid methods and their applications in drug discovery. Trends in pharmacological sciences. 2012. PubMed 22130009
• Felgueiras J, Silva JV, Fardilha M. Adding biological meaning to human protein-protein interactions identified by yeast two-hybrid screenings: A guide through bioinformatics tools. Journal of proteomics. 2018. PubMed 28526529
• Chen Q, Wei T. Membrane and Nuclear Yeast Two-Hybrid Systems. Methods in molecular biology (Clifton, N.J.). 2022. PubMed 34905194
• Reece-Hoyes JS, Walhout AJM. Generating Yeast Two-Hybrid Bait Strains. Cold Spring Harbor protocols. 2018. PubMed 29967272

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