# Yeast Two-Hybrid Screening: Principles and Applications

## Introduction to Yeast Two-Hybrid Screening

### What is Yeast Two-Hybrid Screening?

Yeast two-hybrid screening (Y2H) is a molecular genetic technique used to detect physical interactions between two proteins *in vivo* within the nucleus of *Saccharomyces cerevisiae* (budding yeast). The method exploits the modular architecture of eukaryotic [transcription factors](/knowledge/molecular-biology/transcription-factor) to convert a protein–protein interaction into a quantifiable transcriptional readout—typically the expression of a reporter gene that confers a selectable phenotype or produces a colorimetric signal.

The system was pioneered by Stanley Fields and Ok-Kyu Song in 1989, building on earlier work by Mark Ptashne and colleagues who demonstrated that the DNA-binding and activation functions of the yeast Gal4 [transcription factor](/knowledge/molecular-biology/transcription-factor) reside in separable domains. Since its inception, Y2H has become one of the most widely used tools in [molecular biology](/blog/careers/molecular-biology), enabling genome-wide interaction mapping, discovery of novel binding partners, and functional annotation of uncharacterized proteins. The [Yeast Two Hybrid System](/knowledge/molecular-biology/yeast-two-hybrid-system) remains a cornerstone of interactomics, despite the emergence of complementary technologies.

### Why Study Protein Interactions?

Proteins rarely act in isolation. Cellular processes—[signal transduction](/knowledge/molecular-biology/signal-transduction), transcriptional regulation, DNA replication, vesicular trafficking, and apoptosis—depend on highly coordinated networks of physical protein–protein interactions (PPIs). Identifying these interactions provides critical insight into:

- The biological function of uncharacterized proteins (guilt-by-association).
- The architecture of signaling pathways and multi-protein complexes.
- The molecular basis of disease, where aberrant PPIs often underlie pathology.
- Potential targets for therapeutic intervention, particularly in cases where disrupting a specific interaction is desirable.

Y2H offers a unique advantage: it detects interactions in a living eukaryotic cell, providing a physiologically relevant context (albeit in yeast, not the native organism) while remaining scalable to high-throughput formats. This combination of biological relevance and throughput explains its enduring popularity.

## The Principle Behind the Two-Hybrid System

### The Gal4 Transcription Factor

The *Saccharomyces cerevisiae* Gal4 protein is a transcriptional activator required for the expression of genes involved in galactose metabolism. Gal4 is a modular protein with two functionally distinct domains:

1. **DNA-binding domain (BD)**: Located at the N-terminus (amino acids 1–147), this domain binds specifically to upstream activating sequences (UAS) in the promoter regions of GAL genes.
2. **Activation domain (AD)**: Located at the C-terminus (amino acids 768–881), this domain recruits the transcriptional machinery, including RNA polymerase II and general transcription factors, to initiate transcription.

Critically, these two domains function independently. The BD can bind DNA without activating transcription, and the AD can activate transcription when tethered to DNA, even if it is not covalently linked to the BD. This modularity is the conceptual foundation of the two-hybrid system.

### Bait and Prey Fusion Proteins

The Y2H strategy involves engineering two fusion proteins:

- **Bait**: A protein of interest (protein X) fused to the Gal4 DNA-binding domain (Gal4-BD). The bait is expressed in yeast cells and localizes to the nucleus, where it binds to the UAS in the reporter gene promoter. By itself, the bait cannot activate transcription because it lacks an activation domain.
- **Prey**: A candidate interacting protein (protein Y) fused to the Gal4 activation domain (Gal4-AD). The prey is expressed in the same cell. On its own, the prey cannot bind DNA and therefore cannot activate transcription.

When the bait and prey physically interact, the Gal4-AD is brought into proximity with the UAS-bound Gal4-BD. This reconstitutes a functional transcription factor, which then drives expression of downstream reporter genes. The readout—cell growth on selective media, blue colony color, or fluorescence—indicates a positive interaction.

The system is conceptually elegant: it converts a bimolecular interaction into a unimolecular transcriptional event, amplifying the signal through multiple rounds of reporter mRNA synthesis. This amplification contributes to the sensitivity of the assay, allowing detection of relatively weak or transient interactions that might be missed by biochemical methods. For a more detailed walkthrough of the mechanics, see the [Yeast Two Hybrid Assay](/knowledge/molecular-biology/yeast-two-hybrid-assay).

## Key Components and Vectors

### Bait Vector and Prey Vector

Y2H experiments require two distinct plasmid vectors, each carrying a different selectable marker to maintain both plasmids in the same yeast cell.

**Bait vector (e.g., pGBKT7, pGBT9)**: Contains:
- A yeast promoter (typically the constitutive *ADH1* promoter) driving expression of the bait fusion.
- The coding sequence for the Gal4 DNA-binding domain (amino acids 1–147).
- A [multiple cloning site](/knowledge/diagnostics/molecular/multiple-cloning-site-plasmids-structure-function) (MCS) downstream of the BD for in-frame insertion of the bait gene.
- A yeast selectable marker, usually *TRP1*, which allows growth on media lacking tryptophan.
- An *E. coli* origin of replication and antibiotic resistance gene (e.g., ampicillin) for plasmid propagation in bacteria.

**Prey vector (e.g., pGADT7, pGAD424)**: Contains:
- The same *ADH1* promoter.
- The coding sequence for the Gal4 activation domain (amino acids 768–881).
- A MCS for insertion of the prey gene.
- A different yeast selectable marker, typically *LEU2*, allowing growth on media lacking leucine.
- Bacterial elements for propagation.

The use of different auxotrophic markers (e.g., *TRP1* and *LEU2*) ensures that only yeast cells harboring both plasmids can grow on media lacking both tryptophan and leucine (SD/-Trp/-Leu). This double selection is the first step in any Y2H screen.

### Reporter Genes and Selection Markers

Reporter genes are the output of the Y2H system. A typical Y2H strain contains multiple reporter genes, each under the control of a promoter containing Gal4 UAS elements. The most common reporters are:

| Reporter Gene | Readout | Selection Stringency |
|---|---|---|
| *lacZ* (from *E. coli*) | β-galactosidase activity; blue colonies on X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) | Qualitative; visual |
| *HIS3* | Histidine biosynthesis; growth on media lacking histidine | Moderate; requires 3-AT (3-amino-1,2,4-triazole) to suppress background |
| *ADE2* | Adenine biosynthesis; growth on media lacking adenine; white/red colony color | High stringency |
| *MEL1* | α-galactosidase secretion; blue/white on X-α-gal | Moderate; chromogenic |

The *HIS3* reporter is particularly useful because its stringency can be titrated. The *HIS3* gene product (imidazoleglycerol-phosphate dehydratase) is inhibited by 3-AT, a competitive inhibitor. By adding increasing concentrations of 3-AT (typically 1–100 mM) to the selection media, researchers can suppress weak background activation and select for stronger interactions.

The *ADE2* reporter offers an additional visual readout: yeast colonies that are *ADE2*-negative accumulate a red pigment in their vacuoles. Thus, on low-adenine media, colonies expressing *ADE2* appear white, while non-expressing colonies are red. This provides a convenient way to distinguish true positives from background.

Common Y2H yeast strains include AH109 and Y187. AH109 carries integrated copies of *lacZ*, *HIS3*, and *ADE2* reporters, while Y187 carries *lacZ* and *HIS3*. These strains also harbor mutations in *GAL4* and *GAL80* (a repressor of Gal4) to eliminate endogenous Gal4 activity that could interfere with the assay. The [Yeast Model Organism](/knowledge/molecular-biology/yeast-model-organism) is ideally suited for this purpose due to its well-characterized genetics, rapid growth, and ease of transformation.

## Step-by-Step Workflow of a Yeast Two-Hybrid Screen

### Library Construction

A Y2H screen can be performed in two modes: a **directed** (one-to-one) test, where a single bait is tested against a single prey, or a **library screen**, where a bait is screened against a large library of potential interactors.

For a library screen, the prey library is constructed by cloning cDNA fragments (or open reading frames) from the organism or tissue of interest into the prey vector. Libraries typically contain 10⁵–10⁷ independent clones, ensuring comprehensive coverage of the transcriptome. The cDNA is inserted downstream of the Gal4-AD in all three reading frames to maximize the chance of producing in-frame fusions. Libraries can be:

- **Random-primed**: cDNA fragments generated using random hexamer primers, producing fragments of varying sizes (typically 0.5–3 kb).
- **Oligo(dT)-primed**: Full-length or near-full-length cDNAs generated from poly(A)⁺ mRNA.

The bait plasmid is constructed by cloning the full-length coding sequence of the protein of interest into the bait vector, again ensuring an in-frame fusion with Gal4-BD.

### Screening and Selection

The workflow proceeds as follows:

1. **Transform the bait plasmid** into a haploid yeast strain (e.g., AH109, mating type *MATa*). Plate on SD/-Trp to select for transformants.
2. **Transform the prey library** into a haploid strain of the opposite mating type (e.g., Y187, mating type *MATα*). Plate on SD/-Leu to select for library transformants. The number of transformants should exceed the library complexity by at least 3-fold to ensure representation.
3. **Mate the two strains**: Combine the bait strain and prey library strain in rich medium (YPD) and incubate at 30°C for 4–6 hours with gentle agitation. During mating, diploid cells form that contain both plasmids.
4. **Plate the diploids** on high-stringency selection media, typically SD/-Trp/-Leu/-His/-Ade, supplemented with 3-AT if needed. Only diploids expressing interacting bait–prey pairs will grow.
5. **Incubate at 30°C for 3–7 days**, monitoring for colony appearance. Colonies that appear are candidate positives.
6. **Test for β-galactosidase activity** (if *lacZ* is a reporter) by colony-lift assay or liquid assay using ONPG (o-nitrophenyl-β-D-galactopyranoside) as substrate. This confirms transcriptional activation.

An alternative to mating is **co-transformation**, where both bait and prey plasmids are introduced simultaneously into the same yeast strain. This is simpler but less efficient for library-scale screens.

### Confirming Interactions

Positive colonies from the primary screen must be validated to eliminate false positives:

1. **Isolate prey plasmids** from candidate yeast colonies by plasmid rescue: yeast cells are lysed (e.g., by glass bead disruption or enzymatic digestion with zymolyase), and the plasmid DNA is transformed into *E. coli*. The prey plasmid is selected on ampicillin-containing media, while the bait plasmid (which carries a different bacterial resistance, e.g., kanamycin) is counterselected.
2. **Sequence the prey insert** to identify the interacting protein.
3. **Retest the interaction** by re-transforming the isolated prey plasmid into fresh yeast cells containing the original bait. This confirms that the interaction is reproducible and not an artifact of the initial screen.
4. **Test for specificity**: The prey should not interact with an unrelated bait (e.g., lamin C, a commonly used negative control) or with the Gal4-BD alone. This rules out non-specific binding to the BD or to the UAS.
5. **Perform reciprocal testing**: Swap the bait and prey (i.e., fuse the prey to BD and the bait to AD) and retest. If the interaction is genuine, it should be detectable regardless of fusion orientation, although some interactions are orientation-dependent due to steric constraints.

For a comprehensive overview of the entire procedure, the [Yeast Two-hybrid Y2h](/knowledge/molecular-biology/yeast-two-hybrid-y2h) resource provides additional practical guidance.

## Applications of Yeast Two-Hybrid Screening

### Interaction Mapping

Y2H has been instrumental in generating protein–protein interaction networks for numerous organisms, from yeast and worms to humans. Large-scale screens have produced interactome maps that serve as frameworks for understanding cellular function. For example:

- The yeast interactome, comprising thousands of interactions, has been mapped through multiple independent Y2H screens.
- Human interactome projects have identified tens of thousands of PPIs, many involving disease-associated proteins.
- Viral–host interaction maps have revealed how pathogens hijack host cellular machinery.

These maps are valuable hypothesis-generating tools: a novel interaction between a protein of unknown function and a well-characterized partner immediately suggests a functional link.

### Disease Research

Y2H has contributed significantly to understanding the molecular basis of human diseases:

- **Huntington's disease**: Y2H screens identified huntingtin-interacting proteins, revealing pathways involved in neurodegeneration.
- **Cancer**: Screens with tumor suppressors (e.g., p53, Rb) and oncoproteins (e.g., Myc, Ras) have identified novel binding partners that modulate [cell proliferation](/blog/guides/cell-proliferation) and apoptosis.
- **Infectious disease**: Screens with HIV proteins (e.g., Tat, Nef, Vpr) have uncovered host factors that facilitate or restrict viral replication.
- **Inherited disorders**: Y2H has been used to map interactions of proteins mutated in cystic fibrosis, muscular dystrophy, and other genetic conditions, providing insight into pathogenic mechanisms.

In drug discovery, Y2H can be adapted to screen for small molecules that disrupt or enhance a specific PPI. This is typically done using a **reverse two-hybrid** approach, where interaction is required for cell death (e.g., via expression of a toxic reporter such as *URA3* in the presence of 5-fluoroorotic acid). Compounds that disrupt the interaction allow cell survival, providing a positive selection for inhibitors.

## Advantages and Limitations

### Advantages

- **In vivo context**: Interactions occur inside a living eukaryotic cell, allowing proper protein folding and, in some cases, post-translational modifications.
- **Sensitivity**: The transcriptional amplification step allows detection of weak or transient interactions (dissociation constants in the low micromolar range).
- **Scalability**: Library screens can interrogate millions of potential interactions simultaneously.
- **Simplicity and cost-effectiveness**: Requires only basic [molecular biology](/blog/careers/molecular-biology) equipment; no specialized instrumentation (e.g., mass spectrometry) is needed.
- **Applicability to any organism**: cDNA libraries can be constructed from any source, enabling study of non-model organisms.
- **Detection of binary interactions**: Unlike co-immunoprecipitation (co-IP), which detects complexes, Y2H directly reports a physical interaction between two specific proteins.

### Limitations

- **False positives**: Interactions detected in Y2H may not occur in the native organism due to differences in protein concentration, localization, or post-translational modifications. Some proteins are "sticky" and interact non-specifically with many partners.
- **False negatives**: Interactions requiring post-translational modifications absent in yeast (e.g., specific phosphorylation by mammalian kinases) will be missed. Membrane proteins and proteins with strong hydrophobic regions often fail to fold properly or fail to localize to the nucleus.
- **Nuclear requirement**: Both fusion proteins must enter the nucleus for the assay to work. Proteins with strong nuclear export signals or transmembrane domains are problematic.
- **Toxicity**: Overexpression of certain fusion proteins can be toxic to yeast, preventing growth and yielding false negatives.
- **Autoactivation**: Some baits can activate transcription on their own, without a prey partner, producing high background.

## Common Pitfalls and Troubleshooting

### Autoactivation of Bait

A frequent problem is **autoactivation**, where the bait fusion protein activates reporter gene expression in the absence of a prey. This occurs when the bait protein contains an intrinsic transcriptional activation domain or when the Gal4-BD itself has residual activation activity at high expression levels.

**Troubleshooting**:
- Test the bait alone on selection media lacking histidine and adenine. If colonies grow, the bait is autoactivating.
- Increase the concentration of 3-AT in the media (up to 100 mM) to suppress weak autoactivation.
- Use a less sensitive reporter combination (e.g., *ADE2* alone, which is more stringent than *HIS3*).
- Truncate the bait protein to remove the activation domain, provided the interaction interface is retained.
- Reduce bait expression by using a weaker promoter or a lower-copy plasmid.

### False Positives and Negatives

**False positives** arise from:
- "Sticky" proteins (e.g., highly charged or hydrophobic proteins) that bind non-specifically.
- Interactions that occur only because both proteins are artificially concentrated in the nucleus.
- Chaperone-mediated interactions that do not occur in the native context.

**Mitigation**:
- Use multiple reporter genes; true positives should activate all reporters.
- Include negative controls (e.g., lamin C, empty BD vector).
- Verify interactions using an orthogonal method such as co-IP or FRET.

**False negatives** arise from:
- Poor expression of fusion proteins (check by Western blot).
- Failure of the fusion protein to fold correctly.
- Inability of the protein to enter the nucleus.
- Post-translational modifications required for interaction but absent in yeast.

**Mitigation**:
- Confirm protein expression by Western blot using antibodies against the Gal4-BD or Gal4-AD epitopes.
- Use a different vector system (e.g., the LexA system, which uses the bacterial LexA DNA-binding domain instead of Gal4-BD, reducing autoactivation issues).
- Consider alternative Y2H variants, such as the [Yeast 3 Hybrid System](/knowledge/molecular-biology/yeast-3-hybrid-system), for interactions requiring a third component (e.g., RNA-mediated interactions).

### Weak Interactions

Weak or transient interactions may produce slow growth or faint blue color, making them difficult to distinguish from background.

**Troubleshooting**:
- Increase incubation time (up to 14 days).
- Use lower concentrations of 3-AT to reduce suppression of weak signals.
- Optimize the bait/prey expression ratio by using different promoter strengths.
- Perform the screen at lower temperature (e.g., 25°C instead of 30°C) to stabilize weak interactions.

### Toxicity of Fusion Proteins

Some bait or prey proteins are toxic to yeast, causing slow growth or cell death.

**Troubleshooting**:
- Use an inducible promoter (e.g., *GAL1* promoter) to control expression timing.
- Use a low-copy (centromeric) plasmid to reduce expression levels.
- Screen for transformants on rich media before transferring to selective media.

## Alternative and Complementary Methods

### Co-Immunoprecipitation

Co-immunoprecipitation (co-IP) is the most common orthogonal validation method for Y2H results. In co-IP, a protein of interest is immunoprecipitated from cell lysates using an antibody, and any associated proteins are detected by Western blotting with antibodies against the suspected partner. Unlike Y2H, co-IP occurs in the native cellular context, preserving post-translational modifications and subcellular localization. However, co-IP detects stable complexes, not necessarily direct binary interactions, and requires high-quality antibodies or epitope tags.

### Membrane Yeast Two-Hybrid

The **membrane yeast two-hybrid (MYTH)** system, also known as the split-ubiquitin assay, extends Y2H to membrane proteins. In MYTH, the bait is fused to the C-terminal half of ubiquitin (Cub) and the prey to the N-terminal half (Nub). When the proteins interact, the ubiquitin halves reassemble, triggering cleavage by ubiquitin-specific proteases and release of a transcription factor that activates reporter genes. This system allows detection of interactions involving integral membrane proteins, which cannot be studied by conventional Y2H. The [Yeast Two-hybridization](/knowledge/molecular-biology/yeast-two-hybridization) approach encompasses both classical and split-ubiquitin variants.

### Fluorescence Resonance Energy Transfer (FRET)

FRET measures the distance-dependent transfer of energy between two fluorophores attached to proteins of interest. When the proteins interact (within 10 nm), excitation of the donor fluorophore leads to emission from the acceptor. FRET can be measured in living cells by microscopy or flow cytometry, providing spatial and temporal information about interactions. It is particularly useful for confirming interactions in mammalian cells and for studying dynamic changes in interactions.

### Other Two-Hybrid Variants

- **Yeast three-hybrid (Y3H)**: Detects RNA–protein interactions by using a hybrid RNA molecule that bridges the BD and AD fusions. The [Yeast 3 Hybrid System](/knowledge/molecular-biology/yeast-3-hybrid-system) is also used for interactions requiring a third protein component.
- **Bacterial two-hybrid**: Uses a bacterial transcription factor (e.g., the adenylate cyclase system) to detect interactions in *E. coli*, offering faster growth and different post-translational modification contexts.
- **Split-luciferase**: Similar to MYTH but uses split luciferase fragments; interaction reconstitutes luciferase activity, producing a quantitative luminescent signal.

## Summary and Key Takeaways

Yeast two-hybrid screening remains a powerful and versatile method for detecting protein–protein interactions. Its conceptual simplicity—splitting a transcription factor into two domains and reconstituting activity through protein interaction—belies its profound impact on molecular biology. From genome-wide interactome mapping to targeted studies of disease mechanisms, Y2H has enabled discoveries that would be difficult or impossible with biochemical methods alone.

The technique is not without challenges. False positives and negatives, autoactivation, and limitations with membrane proteins require careful experimental design and orthogonal validation. However, when used appropriately and interpreted critically, Y2H provides invaluable insights into the molecular networks that govern cellular function.

## Frequently Asked Questions

### What is yeast two-hybrid screening?

Yeast two-hybrid screening (Y2H) is a molecular biology technique used to detect physical interactions between two proteins. It is performed in *Saccharomyces cerevisiae* and relies on the modular nature of the Gal4 transcription factor. One protein (bait) is fused to a DNA-binding domain, and another (prey) is fused to an activation domain. If the proteins interact, the two domains are brought together, reconstituting a functional transcription factor that activates reporter gene expression.

### How does yeast two-hybrid screening work?

The bait protein is fused to the Gal4 DNA-binding domain (Gal4-BD), which binds to upstream activating sequences in reporter gene promoters. The prey protein is fused to the Gal4 activation domain (Gal4-AD). When bait and prey interact, the AD is recruited to the promoter, activating transcription of reporter genes such as *HIS3*, *ADE2*, or *lacZ*. This produces a selectable phenotype (growth on media lacking histidine or adenine) or a colorimetric signal (blue colonies on X-gal).

### What are bait and prey in yeast two-hybrid?

The **bait** is the protein of interest fused to the Gal4 DNA-binding domain. It serves as the "hook" that binds to the promoter. The **prey** is a candidate interacting protein fused to the Gal4 activation domain. In a library screen, the prey is typically a collection of cDNA clones representing many potential interactors. The bait is the known protein, while the prey is the unknown.

### What are common reporter genes used in yeast two-hybrid?

Common reporter genes include:
- ***lacZ***: Encodes β-galactosidase; produces blue colonies on X-gal.
- ***HIS3***: Encodes imidazoleglycerol-phosphate dehydratase; allows growth on media lacking histidine. Stringency can be increased with 3-AT.
- ***ADE2***: Encodes phosphoribosylaminoimidazole carboxylase; allows growth on media lacking adenine and produces white colonies (vs. red for non-expressing cells).
- ***MEL1***: Encodes α-galactosidase; produces blue colonies on X-α-gal.

### Why do yeast two-hybrid screens produce false positives?

False positives occur when reporter gene activation is detected without a genuine biological interaction. Causes include: (1) "sticky" proteins that bind non-specifically to many partners; (2) autoactivation, where the bait activates transcription on its own; (3) overexpression artifacts, where high local concentrations force weak interactions; and (4) interactions that occur only in the yeast nucleus but not in the native cellular context. Multiple reporter genes and orthogonal validation (e.g., co-IP) help eliminate false positives.

### Can yeast two-hybrid detect interactions with membrane proteins?

Conventional Y2H is generally unsuitable for integral membrane proteins because they cannot fold properly in the nucleus and often fail to localize there. However, the **membrane yeast two-hybrid (MYTH)** system, based on split-ubiquitin, can detect interactions involving membrane proteins. In MYTH, interaction between membrane-bound bait and prey reconstitutes ubiquitin, triggering proteolytic cleavage and release of a transcription factor that activates reporters.

### What is the difference between yeast two-hybrid and co-immunoprecipitation?

Y2H detects direct binary protein–protein interactions in the yeast nucleus, using transcriptional activation as a readout. It is highly sensitive and scalable but can produce false positives and misses interactions requiring post-translational modifications absent in yeast. Co-immunoprecipitation (co-IP) detects protein complexes from cell lysates in the native cellular context, preserving modifications and localization. However, co-IP requires antibodies or epitope tags, detects stable complexes rather than direct binary interactions, and is less amenable to high-throughput screening. The two methods are complementary and are often used together for validation.

## Key Takeaways

- Yeast two-hybrid screening detects direct physical interactions between two proteins by reconstituting a split transcription factor in yeast.
- The bait is fused to a DNA-binding domain; the prey is fused to an activation domain; interaction drives reporter gene expression.
- Key components include bait and prey plasmids with different selectable markers, yeast strains with integrated reporters (*lacZ*, *HIS3*, *ADE2*), and selective growth media.
- The workflow involves constructing bait and prey libraries, transforming or mating yeast strains, selecting on high-stringency media, and confirming interactions by plasmid rescue and retesting.
- Y2H is used for interaction network mapping, disease mechanism studies, and drug target discovery.
- Major limitations include false positives/negatives, autoactivation, and incompatibility with membrane proteins.
- Validation with orthogonal methods such as co-immunoprecipitation or FRET is essential for reliable conclusions.
- Alternative systems, including membrane Y2H (split-ubiquitin) and yeast three-hybrid, extend the utility of the approach to challenging protein classes.

## Further Reading

- Koegl M, Uetz P. *Improving yeast two-hybrid screening systems*. Briefings in [functional genomics](/blog/guides/functional-genomics) & proteomics. 2007. [PubMed 18218650](https://doi.org/10.1093/bfgp/elm035)
- 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](https://doi.org/10.1016/j.jprot.2017.05.012)
- Wallach D et al. *The yeast two-hybrid screening technique and its use in the study of protein-protein interactions in apoptosis*. Current opinion in immunology. 1998. [PubMed 9602300](https://doi.org/10.1016/s0952-7915(98)80240-9)
- Maple J, Møller SG. *Yeast two-hybrid screening*. Methods in molecular biology (Clifton, N.J.). 2007. [PubMed 17417012](https://doi.org/10.1007/978-1-59745-257-1_15)
- Elmore JM, Velásquez-Zapata V, Wise RP. *Next-Generation Yeast Two-Hybrid Screening to Discover Protein-Protein Interactions*. Methods in molecular biology (Clifton, N.J.). 2023. [PubMed 37450150](https://doi.org/10.1007/978-1-0716-3327-4_19)
- McLellan H, Armstrong MR, Birch PRJ. *Yeast Two-Hybrid Screening for Identification of Protein-Protein Interactions in Solanum tuberosum*. Methods in molecular biology (Clifton, N.J.). 2021. [PubMed 34448156](https://doi.org/10.1007/978-1-0716-1609-3_4)

## Related Clinical & Scientific Guides

* [MAPK Pathway: Mechanism, Function, and Clinical Relevance](/knowledge/molecular-biology/mapk-pathway)
* [Mammalian Cell Culture Bioreactors: A Practical Guide](/knowledge/molecular-biology/mammalian-cell-culture-bioreactor)
* [Nucleotide Formation: Biosynthesis and Assembly of DNA/RNA Building Blocks](/knowledge/molecular-biology/nucleotide-formation)