Yeast Two-Hybrid Assay to Determine Protein Self-Association in Yeast Cells

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

1. 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.
2. 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.
3. 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 carr....

Materials

List of materials used in this article

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