Overview
This video demonstrates the yeast two-hybrid assay to detect specific protein self-interactions. In this assay, the protein of interest is tagged to two domains of a transcription factor that encodes the β-galactosidase enzyme. When the two proteins interact, this causes transcription and translation to form the enzyme, which is then detected using a biochemical assay.
Protocol
1. Yeast Transformation
- Harvest yeast by centrifugation (in 50 ml tubes) at 1,500 x g for 5 min at RT. Discard the supernatant, resuspend each pellet in 5 ml of sterile H2O, and pool together.
- Re-centrifuge at 1,500 x g for 5 min at RT and discard the supernatant. Resuspend yeast pellet in 1 ml of freshly prepared, sterile 1x LiAc/TE.
NOTE: Use competent yeast cells within 1 hr of preparation. - Prepare plasmid samples (in 1.5 ml tubes) by adding 200 ng of plasmid DNA for single transformations, or 0.5 - 1 µg of each plasmid DNA for co-transformations, and 100 µg of herring testes carrier DNA (boiled for 20 min and cooled on ice just prior to use).
NOTE: Include a positive control, e.g., yeast co-transformed with pVA3 (encoding for GAL4 DNA-BD fusion with p53 protein) and pTD1 (encoding for GAL4 AD fusion with SV40 large T antigen). - Add 100 µl of the freshly prepared, competent yeast suspension and 600 µl of 1x LiAc/PEG solution (8 ml of stock PEG 3350, 1 ml of stock TE, 1 ml of stock LiAc), and vortex for ~30 sec. Incubate at 30 °C for 30 min with shaking at 200 rpm.
- Add 80 µl of dimethyl sulfoxide (10% v/v final concentration) and mix well by gentle inversion. Heat shock for 15 min in a 42 °C water bath while mixing every 2 - 3 min.
- Chill cell suspension on ice for 2 min and centrifuge at 14,000 x g for 15 sec at RT to recover yeast.
- Resuspend cell pellet in 100 µl of 1x TE and plate on appropriate minimal SD medium plates for selective growth (medium lacking leucine and tryptophan to keep selective pressure on both bait and target plasmids).
- Incubate plates upside-down at 30 °C until colonies are ~2 mm in diameter (usually 4 - 5 days). Plates can be stored at 4 °C for 2 - 3 weeks; for longer storage make glycerol stocks and store at -80 °C.
NOTE: Verify that bait and target proteins are expressed in yeast by immunoblotting and that they do not have autonomous reporter gene activation when separately expressed in yeast. - Add 100 µl of the freshly prepared, competent yeast suspension and 600 µl of 1x LiAc/PEG solution (8 ml of stock PEG 3350, 1 ml of stock TE, 1 ml of stock LiAc), and vortex for ~30 sec. Incubate at 30 °C for 30 min with shaking at 200 rpm.
- Add 80 µl of dimethyl sulfoxide (10% v/v final concentration) and mix well by gentle inversion. Heat shock for 15 min in a 42 °C water bath while mixing every 2 - 3 min.
- Chill cell suspension on ice for 2 min and centrifuge at 14,000 x g for 15 sec at RT to recover yeast.
- Resuspend cell pellet in 100 µl of 1x TE and plate on appropriate minimal SD medium plates for selective growth (medium lacking leucine and tryptophan to keep selective pressure on both bait and target plasmids).
- Incubate plates upside-down at 30 °C until colonies are ~2 mm in diameter (usually 4 - 5 days). Plates can be stored at 4 °C for 2 - 3 weeks; for longer storage make glycerol stocks and store at -80 °C.
NOTE: Verify that bait and target proteins are expressed in yeast by immunoblotting and that they do not have autonomous reporter gene activation when separately expressed in yeast.
2. Colony-lift Filter Paper β-Gal Assay
- Prepare the following buffers:
- Prepare a Z-buffer containing 100 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, and 1 mM MgSO4; adjust the pH to 7.4. Sterilize by autoclaving and store at RT.
- Prepare X-Gal buffer by dissolving 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside at 20 mg/ml in N,N-dimethylformamide, and store in the dark at -20 °C. Prepare Z buffer/X-Gal solution. Make buffer prior to use by mixing X-Gal at 0.33 mg/ml and β-mercaptoethanol at 0.27% v/v in the Z-buffer. Use 2.5 ml per sample.
- Add 2.5 ml of freshly prepared Z buffer/X-Gal solution to a clean 100 mm plate and place it inside a cellulose filter paper.
- Place a new filter paper over the surface of the plate with the yeast colonies to be assayed. Gently rub the filter paper onto the plate with forceps and leave for ~5 min for colonies to attach.
NOTE: Process the positive control in parallel, i.e., yeast co-transformed with pVA3 and pTD1. - Lift the filter paper and submerge it (with forceps) into a pool of liquid nitrogen for 30 sec (liquid nitrogen should be handled with care; always wear thick gloves and goggles). Let the frozen filter paper thaw at RT for ~2 min.
- Place the filter paper (colony side up) on top of the pre-soaked filter paper inside the 100 mm plate, and incubate at 30 °C.
- Check periodically (every ~30 min) for the appearance of blue colonies.
NOTE: Weak bait: target interactions may take several hours to produce a positive blue signal (avoid prolonged incubation (>8 hr) that may give false positive results). For best results, use freshly co-transformed colonies, i.e., grown at 30 °C for 4 - 5 days, ~2 mm in diameter.
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Materials
Name | Company | Catalog Number | Comments |
Plate incubator | Hereaus | B6120 | Device |
Orbital shaker incubator | New Brunswick Scientific | INNOVA 4300 | Device |
Spectrophotometer | Perkin Elmer | Lambda Bio+ | Device |
Matchmaker Two-Hybrid System 2 Kit | Takara Clontech | K1604-1 | Contains bait vector pGBKT7, prey vector pACT2 and yeast strain Y190 |
Yeast Nitrogen Base | Sigma-Aldrich | Y0626 | To prepare minimal SD growh medium |
Dropout supplement lacking leucine and tryptophan | Sigma-Aldrich | Y0750 | To prepare minimal SD growh medium |
Dropout supplement lacking leucine, tryptophan, histidine | Sigma-Aldrich | Y2001 | To prepare minimal SD growh medium |
Herring testes carrier DNA | Takara Clontech | 630440 | For yeast transformation |
Whatman filter paper Grade 5 | Sigma-Aldrich | WHA1005070 | For use in colony-lift filter paper β-Gal assay |
X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) | Sigma-Aldrich | B4252 | For use in colony-lift filter paper β-Gal assay |