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.
1. Yeast Transformation
2. Colony-lift Filter Paper β-Gal Assay
The authors have nothing to disclose.
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 |