Dec 04, 2025
E3 decoy library high-throughput yeast two-hybrid screening assay
- Chen-An Chen1,
- Yi-Tsung Tu1,
- Chin-Mei Lee1
- 1Institute of Plant Biology, National Taiwan University, Taipei, 106319, Taiwan
Protocol Citation: Chen-An Chen, Yi-Tsung Tu, Chin-Mei Lee 2025. E3 decoy library high-throughput yeast two-hybrid screening assay. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpm72pgzp/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: December 04, 2025
Last Modified: December 04, 2025
Protocol Integer ID: 234213
Keywords: E3 ligase, ubiquitination, yeast two-hybrid, protein-protein interaction, Arabidopsis, circadian clock regulation in arabidopsi, arabidopsis e3 ubiquitin, mediated circadian clock regulation, arabidopsi, throughput yeast screen, e3 decoy library, throughput yeast, yeast, potential protein, decoy library, protein interaction
Funders Acknowledgements:
MOST
Grant ID: MOST-109-2311-B-002-030-MY2
NSTC
Grant ID: NSTC-111-2311-B-002-012-MY3
NTU
Grant ID: NTU-CC-112L891804
NTU
Grant ID: NTU-CC-114L895003
MOST
Grant ID: MOST-110-2811-B-002-523-
NSTC
Grant ID: NSTC-113-2811-B-002-122-
NTU
Grant ID: NTU-CC-111L4000
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Abstract
This protocol describes a yeast two-hybrid (Y2H) assay designed to systematically screen for potential protein–protein interactions using the Arabidopsis E3 ubiquitin ligase decoy library. It serves as a detailed methodological supplement to “Dissecting ubiquitin-mediated circadian clock regulation in Arabidopsis via high-throughput yeast screens” by Tu and Chen (2026) (link).
Materials
Yeast strains and vectors
- The opposite mating type of Saccharomyces cerevisiae strains AH109 and Y187.
- The yeast two-hybrid vector pDEST22 serves as the bait vector carrying the gene of interest.
Equipment and consumables
Shaking incubator, cell density meter, 96-well PCR plate, adhesive film for 96-well PCR plate, reagent reservoir, 8-channel pipette, sterile glass culture tube
Troubleshooting
Media recipes
YPD(A) medium
| Components | Amounts | |
| Difco peptone | 20g | |
| Yeast extract | 10g | |
| Dextrose | 20g | |
| Adenine (A) | 0.20g | |
| Total | 1L | |
| Adjust the pH to 6.5 | ||
SD medium
| Components | Amounts | |
| Yeast nitrogen base without amino acids | 6.7g | |
| Dextrose | 20g | |
| Amino acid drop-out mix | 1.5g | |
| Agar (for plates only) | 20g | |
| Total | 1L | |
| Adjust the pH to 5.8 | ||
Amino acid drop-out mix
| Reagent (g) | -Leu | -Trp | -Leu-Trp | -Leu-Trp-His | Brand (catalog no.) | |
| Adenine | 1 | 0.5 | 2 | 0.5 | Bioshop (ADA001.25) | |
| Alanine | 4 | 2 | 8 | 2 | Panreac (A3690) | |
| Arginine | 4 | 2 | 8 | 2 | Panreac (A3675) | |
| Asparagine | 4 | 2 | 8 | 2 | Panreac (A3720) | |
| Aspartic acid | 4 | 2 | 8 | 2 | Sigma (A-7219) | |
| Cysteine | 4 | 2 | 8 | 2 | Panreac (A1703) | |
| Glutamine | 4 | 2 | 8 | 2 | Sigma (G3126) | |
| Glutamic acid | 4 | 2 | 8 | 2 | ACROS (G0059) | |
| Glycine | 4 | 2 | 8 | 2 | Sigma (G-8790) | |
| Histidine | 4 | 2 | 8 | 0 | Panreac (A3733) | |
| Inositol | 4 | 2 | 8 | 2 | Sigma (I-7508) | |
| Isoleucine | 4 | 2 | 8 | 2 | Bioshop (ISO910) | |
| Leucine | 0 | 10 | 0 | 0 | Sigma (L-1512) | |
| Lysine | 4 | 2 | 8 | 2 | Bioshop (LYS101.100) | |
| Methionine | 4 | 2 | 8 | 2 | Panreac (A1340,0100) | |
| Phenylalanine | 4 | 2 | 8 | 2 | Scharlau (FE0180) | |
| Proline | 4 | 2 | 8 | 2 | Scharlau (PR0055) | |
| Serine | 4 | 2 | 8 | 2 | SIGMA (S5386) | |
| Threonine | 4 | 2 | 8 | 2 | Panreac (A3969) | |
| Tryptophan | 4 | 0 | 0 | 0 | Sigma (T-8941) | |
| Tyrosine | 4 | 2 | 8 | 2 | Scharlau (TI0325) | |
| Uracil | 4 | 2 | 8 | 2 | Sigma (U-1128) | |
| Valine | 4 | 2 | 8 | 2 | Scharlau (VA0055) |
Yeast strains and vectors
1. The opposite mating type of Saccharomyces cerevisiae strains AH109 and Y187.
2. The yeast two-hybrid vector pDEST22 serves as the bait vector carrying the gene of interest.
Equipment and consumables
Shaking incubator, cell density meter, 96-well PCR plate, adhesive film for 96-well PCR plate, reagent reservoir, 8-channel pipette, sterile glass culture tube
Procedure
Transformation
Transform pDEST32-E3 decoy into yeast AH109 strain and culture in 3~5mL SD-Leu liquid medium at 30°C for one day.
Transform pDEST22-bait into yeast Y187 strain and culture in 3~5mL SD-Trp liquid medium at 30°C for one day.
Prepare the E3 decoy yeast into 96-well plate
Add 50μl pDEST32-E3 decoy bacterial liquid culture to 100μl SD-Leu liquid medium per well in 96-well plate.
Seal film carefully and ensure that no bacterial liquid is attracted to the film by static electricity.
Incubate at 30°C overnight.
(optional) Drop the bacterial culture on SD-Leu agar plate (2.5μl/drop) and incubate at 30°C for two days. This step allows the E3-decoy yeast cultures to be directly inoculated into liquid medium from the colonies in the next experiment.
Mate the E3 decoy strain and the bait strain
Mix the pDEST32-E3 decoy/pDEST32 culture and the pDEST22-bait/pDEST22 culture in a new 96-well plate. Each well should contain: 30μl pDEST22-bait/pDEST22 culture, 30μl pDEST32-E3 decoy/pDEST32 culture, and 100μl YPDA medium. Notice:
Notice: Centrifuge the pDEST32–E3 decoy 96-well plate at 2000 rpm for 2 minutes before opening it to avoid cross-contamination between wells.
Notice: Pre-mixing the pDEST22-bait/pDEST22 culture with YPDA medium prior to dispensing into the 96-well plate.
Seal film carefully and ensure that no bacterial liquid is attracted to the film by static electricity.
Incubate at 30°C overnight.
Drop 2.5 μl/well mating bacterial culture on SD-Leu-Trp and SD-Leu-Trp-His agar plate.
Check the plate and take photos every day, paying attention on day 3 (for SD-Leu-Trp), and on day 4 and 5 (for SD-Leu-Trp-His).
Identify the potential E3 candidates by comparing with those of the control plate (pDEST32-E3 decoy + pDEST22).
Y2H assay with serial dilution after high-throughput screening
Culture liquid yeast (pDEST22/pDEST22-bait/pDEST32/pDEST32-E3 decoy) in glass tubes, separately. Incubate at 30°C overnight.
Mixing 100μl pDEST22/pDEST22-bait and 100μl pDEST32/pDEST32-E3 decoy in 3ml YPDA medium for mating. Incubate at 30°C overnight with shaking at 200 rpm.
Take 10–20 µl of the culture and streak for single colonies. Incubate at 30 °C for two days.
Pick a single colony and culture into SD-Leu-Trp liquid medium. Incubate at 30°C overnight with shaking at 200 rpm.
Adjust OD600 to 0.3 with SD-Leu-Trp liquid medium as 1x concentration. Prepare the 10x and 100x dilutions with SD-Leu-Trp liquid medium. Mix thoroughly by pipetting to ensure homogeneity before transferring culture to the respective dilutions.
Dropping 2.5 μl/well liquid yeast onto SD-Leu-Trp and SD-Leu-Trp-His agar plates.
Check the plate and take photos every day, especially on day 3 (for SD-Leu-Trp), and on day 4 and 5 (for SD-Leu-Trp-His).