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 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…

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