Yeast Two-Hybrid Assay to Determine Protein Self-Association in Yeast Cells
Published: May 31, 2023
Abstract
Source: Stanczyk, P. J., et al., Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study. J. Vis. Exp. (2016)
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 2…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Tags
Cite This Article
Yeast Two-Hybrid Assay to Determine Protein Self-Association in Yeast Cells. J. Vis. Exp. (Pending Publication), e21404, doi: (2023).