May 11, 2026
A scalable multiplexed Yeast-two Hybrid (mY2H) assay for protein-to-protein interaction (PPIs) screening
- Chantal Nyirakanani1,
- Kerstin Spirohn-Fitzgerald1,
- Luke Lambourne1,
- Ryan Murray1,
- Tong Hao1,
- Michael A. Calderwood1
- 1Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA
Protocol Citation: Chantal Nyirakanani, Kerstin Spirohn-Fitzgerald, Luke Lambourne, Ryan Murray, Tong Hao, Michael A. Calderwood 2026. A scalable multiplexed Yeast-two Hybrid (mY2H) assay for protein-to-protein interaction (PPIs) screening. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3dz5lk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 26, 2023
Last Modified: May 11, 2026
Protocol Integer ID: 88439
Keywords: Multiplexed Yeast Two-Hybrid, Pooled Assay, PPI Perturbation, Variant Screening, Nanopore Sequencing, Multiplexed Yeast Two-Hybrid, PPI Perturbation, Variant Screening, Pooled Assay, High-Throughput Screening, Nanopore Sequencing, multiplexed yeast, scalable multiplexed yeast, simultaneous assessment of multiple genetic variant, yeast transformation, multiple genetic variant, functional genomic, scale functional genomic, type clone for each target gene, systematic screening of protein, target gene, gateway lr recombination, protein interaction, key procedural steps en masse cloning, pooled entry clone, interactome, specific allele, entry clone
Funders Acknowledgements:
National Human Genome Research Institute (NHGRI)
Grant ID: UM1HG011989
Abstract
This protocol describes a high-throughput multiplexed Yeast Two-Hybrid (mY2H) assay designed for the systematic screening of protein-protein interactions (PPIs). The method enables the simultaneous assessment of multiple genetic variants by pooling variants with a wild-type clone for each target gene.
Key Procedural Steps
- En Masse Cloning: Pooled entry clones are transferred into expression vectors using Gateway LR recombination.
- Yeast Transformation: The resulting expression clones are transformed into haploid S. cerevisiae strains (Y8800 and Y8930).
- Mating: performed in triplicate to ensure data robustness and reproducibility.
- Molecular Readout: Interacting pairs are identified via indexed PCR and Nanopore sequencing, enabling high-resolution mapping of how specific alleles rewire the interactome.
This scalable approach significantly increases throughput, making it an efficient tool for large-scale functional genomics and studies of disease mechanisms.
Protocol materials
MAX Efficiency™ DH5α Competent CellsThermo FisherCatalog #18258012
S-Blocks (24), Deepwell, Deep wellQiagenCatalog #19585
Phusion HF DNA PolymeraseThermo Fisher ScientificCatalog #F530L
E-Gel™ 96 Agarose GelsLife TechnologiesCatalog #G700801
Adenine hemisulfate saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #A-3159
3-Amino-1,2,4-triazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #A8056-100G
Bacteriological Agar, UltrapureThermo FisherCatalog #J10906.Q1
UracilMerck MilliporeSigma (Sigma-Aldrich)Catalog #U0750-500G
D-( )-GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8270-25KG
Yeast Nitrogen Base Without Amino Acids and Ammonium SulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y1251-1KG
L-HistidineMerck MilliporeSigma (Sigma-Aldrich)Catalog #H8000
Ammonium SulfateFisher ScientificCatalog #A702-10
Zymolase, 20TUS BiologicalCatalog #Z1000
PCR plate, 96-well, full skirt, natural, 25/boxMedSupply PartnersCatalog #15-2844
LR Clonase Mix IILife TechnologiesCatalog #11791100
QIAquick PCR Purification Kit (50)QiagenCatalog #28104
QIAquick Gel Extraction Kit – Gel PurificationQiagenCatalog #28704
1. Preparation of Allelic Entry-Clone Pools
30m
Inoculate each entry clone (wild-type and alleles) in 1ml LB-Spec (plasmid backbone: pDONR-223) and incubate overnight at 37 °C 850 rpm
.
The next day, for each gene, a pool of 30 clones comprising 28 allele variants and a wild-type clone was created. To ensure adequate wild-type representation, twice the amount of the wild-type gene was added to the pool. An equal amount of each allele culture was combined to a final pool volume of 2 mL (in a deep well plate).
S-Blocks (24), Deepwell, Deep wellQiagenCatalog #19585
Spin the deep well plate at 4000 rpm 00:30:00 and extract DNA using a 96-well DNA extraction kit.
30m
Normalize DNA to 50ng/ul.
2. En Masse Gateway LR Recombination for the Generation of Expression Clones
Set up En masse LR reactions and incubate overnight at 25 °C in a 96-well PCR plate.
PCR plate, 96-well, full skirt, natural, 25/boxMedSupply PartnersCatalog #15-2844
For one reaction:
LR Clonase Mix IILife TechnologiesCatalog #11791100
| A | B | |
| entry pool (50ng/ul) | 3ul | |
| LR clonase Mix II | 2ul | |
| pDEST (150ng/ul) | 1ul | |
| TE buffer, pH8.0 | 4ul |
The pDEST plasmid varies depending on your assay version (in which orientation and plasmid combination the PPI was found in Luck et al. 2020). Here, we used assay versions 1, 2 and 3 in two orientations.
Vector details
| A | B | C | D | E | |
| Name | pDEST-DB | pDEST-AD-CYH2 | pDEST-QZ213 | pDEST-AD-AR68 | |
| Fusion | Gal4-DB(aa 1-147) | Gal4-AD(aa 768-881) | Gal4-AD(aa 768-881) | Gal4-AD(aa 768-881) | |
| Fusion location | N-term | N-term | N-term | C-term | |
| Promoter | Truncated ADH1 promoter (-701 to +1) | Truncated ADH1 promoter (-701 to +1) | Truncated ADH1 promoter (-410 to +1) | Truncated ADH1 promoter (-410 to +1) | |
| Yeast replication ori | CEN | CEN | 2micron | 2micron | |
| Linker | SRSNQ | GGSNQ | ICMAYPYDVPDYASLGGHMAMEAPS | VDGTA | |
| Terminator | ADH1 Term | ADH1 Term | ADH1 Term | ADH1 Term | |
| Selection marker | AmpR | AmpR | AmpR | AmpR |
Assay versions
| A | B | C | D | E | |
| Assay version | DB vector | AD vector | DB yeast strain | AD yeast strain | |
| 1 | pDEST-DB | pDEST-AD-CYH2 | Y8930 | Y8800 | |
| 2 | pDEST-DB | pDEST-QZ213 | Y8930 | Y8800 | |
| 3 | pDEST-DB | pDEST-AD-AR68 | Y8930 | Y8800 |
3. Transformation of E. coli DH5α with Products of En Masse LR Recombination
1h 47m 45s
- Pre-chill the PCR plate and 50ml basin, turn on the 96-well heating block to 42 °C .
- For each LR reaction: Thaw 50 µL of competent cells On ice .
- MAX Efficiency™ DH5α Competent CellsThermo FisherCatalog #18258012 (or home-made DH5alpha competent cells
- Once competent cells are defrosted, transfer them into the cool basin and pipette 50 µL /well.
- Add 2.5 µL of the LR reaction and incubate 00:15:00 On ice .
- While the cells are incubating, add 500 µL per well of SOC media into a deep well plate.
- After incubation, heat-shock the cells at 42 °C 00:00:45 a and immediately chill On ice for 00:02:00 .
- Transfer all cells into a deep well containing 500 µL e SOC per well and incubate at 37 °C 850 rpm for 01:00:00 .
- Meanwhile, fill a new deep well plate with1200 µL of LB-ampicillin.
- After incubation, transfer 250 µL of the recovery solution into the deep well plate containing 1200 µL LB-amp. In addition, we like to spot 5 µL of the recovery onto a 15cm LB-amp petri dish. This can help indicate the success of the LR reactions and transformation.
- Incubate the deep well at 37 °C 850 rpm overnight. The spotted 15Cm LB-amp petri dish are incubated at 37 °C overnight.
- Next day, make two copies of glycerol stocks (1:1 ratio of 40% glycerol and culture). Store your bacteria expression clones at -80°C.
- Spin the bacteria culture at 4000 rpm for 00:30:00 and perform 96-well DNA purification (following Qiagen protocol).
- Inoculate a single colony of desired haploid S. cerevisiae yeast strains to perform yeast transformations the next day: Y8800 (MATa) for allele/WT pools in pDEST-AD-prey plasmids, Y8930 (MATα) for alleles/WT in DB plasmid (plasmid details see table in step 5) --> pick one colony into ~50 mL YPD media and incubate at 30 °C 200 rpm overnight.
1h 47m 45s
4. Yeast transformation
4h 15m
- Haploid S. cerevisiae strains Y8800 (MATa) and Y8930 (MATα) are transformed with pDest-AD-prey vectors (carrying the TRP1 gene) and pDest-DB-bait vectors (carrying the LEU2 gene), respectively.
- Harvest the yeast culture when it reaches an absorbance of OD600 0.8 to 1.0 (roughly mid-log phase growth). Note: If the absorbance greatly exceeds, adjust the cell density to an OD600 of 0.33 with fresh YPD and grow at 30 °C for 03:00:00 (approximately two doublings).
- Pellet cells at 2000 rpm for 00:05:00 and discard supernatant.
- Wash cells by resuspending the pellet with 10 mL sterile water.
- Spin at 2000 rpm for 00:05:00 . Discard supernatant. OPTIONAL: Wash cells a second time by resuspending the pellet with 5 mL of 0.1M LiAc. Spin at 2000 rpm for 00:05:00 . Discard supernatant.
- Completely resuspend pellet in yeast transformation (TRAFO) buffer:
TRAFO Buffer 1 tfn 50 tfns 100 tfns
60% PEG3350 25 μl 1250 μl 2500 μl
1M LiAc 4.5 μl 225 μl 450 μl
water 9.25 μl 462.5 μl 925 μl
2mg/ml ssDNA 6.25 μl 312.5 μl 625 μl
Total 45 μl 2250 μl 4500 μl
7. Aliquot 45 µL /well of the TRAFO buffer into a 96-well Costar plate (round bottom) and add 5-7 µL of DNA from step 6.
8. Seal plates and incubate them at 42 °C for 01:00:00 .
9. Spot 5-7 µL on selective agar -solid media: SC-L for DB vector and SC-W for AD vector. Incubate at 30 °C for 3days.
10. After 3 days at 30°C, pick colonies into liquid selective media SC-L for DB and SC-W for AD and incubate for 2 days at 30 °C .
11. After incubation, make two copies of glycerol stocks (1:1 ratio of 40% glycerol and culture). Store your yeast expression clones at -80°C until ready for the next step.
4h 15m
5. QC of allelic/wild-type pools
2h 5m
Yeast Lysate Preparation Protocol
Stock solutions:
- 0.2M dibasic sodium phosphate (Fisher Scientific, #S369500) Na2HPO47H2O (MW = 268.07), dissolve 53.65g in 1L ddH2O, adjust pH to 7.4
- 0.2M monobasic sodium phosphate (Fisher Scientific, #S373500) NaH2PO4H2O (MW = 138.01), dissolve 27.6g in 1L ddH2O, adjust pH to 7.4
- For 25 mL Lysate buffer: 0.2M Na2HPO47H2O = 20.25 mL 0.2M NaH2PO4H2O = 4.75 mL
- Make the lysis buffer by combining Na2HPO47H2O and NaH2PO4H2O to a pH of 7.5. After mixing the two buffers, add zymolase: for 1ml lysis buffer = 2mg zymolase
Zymolase, 20TUS BiologicalCatalog #Z1000
- Distribute 25 µL of lysis buffer/well
- Add 6 µL saturated yeast/well
- Seal the plates with the heat sealer and incubate at 37 °C for 02:00:00 (Don’t stack plates after heat sealing them; the lid is too hot).
- Heat shock at 60 °C for 00:05:00
- Add 80 µL of water (using electronic Ovation multichannel pipette)
- Store the plates at -20C and set up indexed PCR for quality control
2h 5m
Indexed PCR
Phusion HF DNA PolymeraseThermo Fisher ScientificCatalog #F530L
| A | B | |
| Components | 1rx (uL) | |
| Phusion 2x | 15 | |
| (0.2uM fc) Term-reverse (2uM stock) | 3 | |
| (0.2uM fc) AD or DB-forward (2uM stock) | 3 | |
| Yeast lysate template | 2 | |
| (4% fc) DMSO (100% stock) | 1.2 | |
| DEPC water | 5.8 | |
| TOTAL | 30 uL |
Gel of PCR products: 96-well Agarose gel following the manufacturers protocol
E-Gel™ 96 Agarose GelsLife TechnologiesCatalog #G700801
PCR products Pooling and Purification
Each well of each PCR plate contains amplicons with two unique barcodes and can be pooled into a single sample. The pooled products were purified by first using the Qiagen PCR purification kit, followed by gel purification (Qiagen gel purification kit). The purified samples were sent to Plasmidsaurus for custom Nanopore sequencing.
QIAquick PCR Purification Kit (50)QiagenCatalog #28104
QIAquick Gel Extraction Kit – Gel PurificationQiagenCatalog #28704
6. mY2H assay
Before starting the assay:
- Pour selective media plates (Corning 245mm Square BioAssay Dishes, cat. 431111) ~ one week before the assay to allow the plates to dry. If on the day of spotting the plates are still 'too wet' (if spots merge), dry the square plates in a cell culture hood.
Preparation of SC-LW solid media (1L):
Bacteriological Agar, UltrapureThermo FisherCatalog #J10906.Q1
UracilMerck MilliporeSigma (Sigma-Aldrich)Catalog #U0750-500G
L-HistidineMerck MilliporeSigma (Sigma-Aldrich)Catalog #H8000
Ammonium SulfateFisher ScientificCatalog #A702-10
D-( )-GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8270-25KG
Adenine hemisulfate saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #A-3159
Yeast Nitrogen Base Without Amino Acids and Ammonium SulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y1251-1KG
| A | B | C | |
| Amino acid mix (lacking Leucine/Tryptophan/Histidin/Uracil) | 1.3 | g | |
| Yeast nitrogen base | 1.7 | g | |
| Ammonium sulfate | 5 | g | |
| H2O | 950 | ml | |
| Agar | 20 | g | |
| pH 5.9, autoclave, cool | |||
| 40% glucose | 50 | ml | |
| 40mM adenine hemisulfate | 12 | ml | |
| 100mM histidine | 8 | ml | |
| 20mM uracil | 8 | ml |
Before adding agar, fully dissolve SC ingredients. Agar will dissolve during autoclaving. Add a magnetic stir bar before autoclaving. After autoclaving, place the flask on a magnetic stirrer to allow the media to cool off before adding the remaining additives (glucose and amino acids).
Preparation ofSC-LWH+1mM3AT (1L):
3-Amino-1,2,4-triazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #A8056-100G
| A | B | C | |
| Amino acid mix (lacking Leucine/Tryptophan/Histidin/Uracil) | 1.3 | g | |
| Yeast nitrogen base | 1.7 | g | |
| Ammonium sulfate | 5 | g | |
| H2O | 950 | ml | |
| Agar | 20 | g | |
| pH 5.9, autoclave, cool | |||
| 40% glucose | 50 | ml | |
| 40mM adenine hemisulfate | 12 | ml | |
| 100mM histidine | 8 | ml | |
| 20mM uracil | 8 | ml | |
| 2M 3-AT (3-Amino-1,2,4-triazole) | 500 | ul |
Before adding agar, fully dissolve SC ingredients. Agar will dissolve during autoclaving. Add a magnetic stir bar before autoclaving. After autoclaving, place the flask on a magnetic stirrer to allow the media to cool off before adding the remaining additives (glucose, amino acids, and 3-AT).
Day 1:
Inoculation of transformed yeast cells
- Inoculate 5 µL of DBs 'glycerol stocks into 200 µL of SC-L medium, and 5 µL of AD glycerol stock into 200 µL of SC-W medium using a 96-well liquid handling robot or a multichannel pipette.
- If the alleles/WT pools are on the DB side (Y8930), inoculate an 'AD-null' strain (Y8800 with the empty pDEST-AD not containing an ORF) to help identify auto-activators.
Note: depending on the assay version, the interactors, either in pDEST-AD or pDEST-DB, need to get cherry picked before the amY2H from the human ORFeome collection 9.1.
- At the same time, inoculate the 6 DB and AD controls in SC-L and SC-W,
- respectively (Dreze et al.).
- Incubate both cultures at 30 °C for 2 days.
- If your experiment involves many samples, a re-array step
- will be performed to organize the DB and AD clones, making the mating process
- easier.
Day3:
Step 2. Mating of Yeast Strains
- Combine equal volumes 5 µL of the DB and AD cultures into 180 µL of YPD to allow mating. Each mating is done in triplicates.
- All DB cultures should also be mated with the 'AD-null' yeast strain
- Perform the mating of control as well.
- Incubate the mixture overnight at 30 °C .
Day 4:
Step 3: Diploid yeast cell enrichment
o Inoculate 180 µL of SC-LW medium with 10 µL of mated cultures.
o Incubate the mixture overnight at 30 °C .
Day 5: Step 4: Spotting on solid media
o SC-LW: This is to select diploid yeast cells.
o SC-LWH+1mM3AT: To screen for interaction
o Incubate at 30 °C for 3 days.
o On the 4th day, leave the plate at RT for overnight
Day 6: Step 5: Take plate pictures, and Plate Scoring
The steps are essentially the same as those used in pairwise Y2H. The key difference in the pooled Y2H approach is that multiple alleles are pooled and screened simultaneously against a single interaction partner, enabling high-throughput assessment of interaction effects. In addition, mating reactions are conducted in triplicate to enhance the robustness and reproducibility of interaction measurements.
After step 5, continue to the following steps:
- Pick colonies from SC-LW and 3AT plates into 180 µL of corresponding liquid media SC-LW or SC-LWH 1Mm3AT.
- Make yeast lysate (see step 8). Keep one copy of glycerol stock as a backup in -80 oC .
- Perform PCR using barcoded primer sets.
- Pool PCR products and purify them
- Follow the sequencing company’s instructions for sample preparation.
- Seq Data Analysis.
Indexed PCR for LW and 3AT plates
| A | B | |
| Components | 1rx (uL) | |
| Phusion 2x | 15 | |
| (0.2uM fc) Term-I5-X (2uM stock) | 3 | |
| (0.2 uM fc) Forward DB-Y (2uM stock) | 3 | |
| Yeast lysate template | 2 | |
| (4% fc) DMSO (100% stock) | 1.2 | |
| DEPC water | 5.8 | |
| TOTAL | 30 uL |
PCR conditions:
98----2min
98------30 sec
57------10 sec 30 cycles
72---------ext time (30 sec/kb)
72-----------10min
12---------hold
7. Sequencing of samples
For each replicate, we perform PCR with different forward and reverse barcoded primer sets (6 different sets in total). Therefore, each PCR plate contains amplicons with two unique barcodes and can now be pooled.
For example, one 96-well plate of amY2H will become 6 plates (3 replicates, two media conditions).
3AT replicates will be pooled with LW replicates, then PCR purified (Qiagen PCR purification kit).
After PCR purification, 1 μg of 3AT and 1 μg of LW samples are pooled and gel-purified (Qiagen gel purification kit). This step is crucial for removing unwanted by-products.
The purified samples were sent to Plasmidsaurus for custom Nanopore sequencing.