"One plate/three-reporter" assay format for the detection and validation of yeast two-hybrid interactions. (November 2004)

• Record Type: Journal Article
• Authors: Evans, David R.H.; Swirsding, Kendra A.; Taillon, Bruce E.; Simons, Jan F.
• Is Part Of: Biotechniques. Volume 37:Number 5(2004)
• Page Start: 840
• Page End: 843
• Publication Date: 2004-11
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/04375PT02
• Languages: English
• ISSNs: 0736-6205

Abstract

We describe a novel assay format for the Gal4-based yeast two-hybrid-system, in which the readout from three different reporter genes is measured sequentially in a single microplate. Activation of the URA3, MEL1, and lacZ reporters in response to a protein-protein interaction is monitored by measuring sequentially: ( i ) growth in medium lacking uracil, ( ii ) α-galactosidase activity, and ( iii ) β-galactosidase. The data thus generated permit elimination of many false positive signals and provide a preliminary measurement of reporter activation-strength that may be confirmed by further analysis. The assay procedure is inexpensive and requires few liquid-handling steps. It is appropriate for automated high-throughput interaction mating assays, validation of putative interactor strains and hybrid-protein self-activator tests.