Protein interaction analysis in yeast-two-hybrid (Y2H) system

Used in Yu et al., Elife. 2022 Apr 11;11:e74531.

1. Transform the Y2H plasmids (pGADT7 and pGBKT7 derived plasmids) into S. cerevisiae strains Y2H gold (Clontech Laboratories) with lithium acetate/polyethylene glycol method developed by Gietz and Woods (Methods Enzymol. 2002; 350:87-96).

2. Inoculate single colonies from yeast transformation plates into 3 ml SD -Leu, -Trp medium, grow the cells on culture rotator at 30 ^oC overnight

3. Inoculate the overnight culture into fresh 3 ml SD -Leu, -Trp medium (OD₆₀₀=0.1), grow the cells on culture rotator at 30 ^oC for 6 hrs to OD₆₀₀ ~0.8

4. Pellet the cells with centrifugation at 3000 × g for 5 min

5. Wash the cells twice with autoclaved water

6. Resuspend the cells in 1 ml autoclaved water, measure the OD₆₀₀ and dilute the cells to OD₆₀₀=0.1

7. Place 10 uL dilution of cells in test (OD₆₀₀=0.1) as one droplet on the selective plates (SD -Leu, -Trp or SD -Leu -Trp, -His, with 10 mM 3-amino-1,2,4-triazole [3-AT])

8. Air-dry the droplets on the plates and seal the plates with parafilm

9. Grow the cells at 30 oC for 2-3 days

10. Take picture of the colonies on the plates to analyze the protein interaction under test