"One plate/three-reporter" assay format for the detection and validation of yeast two-hybrid interactions.
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) alpha-galactosidase activity, and (iii) beta-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.
Evans
David R H
DR
CuraGen Corporation, Branford, CT 06405, USA. devans@curagen.com
Swirsding
Kendra A
KA
Taillon
Bruce E
BE
Simons
Jan F
JF
D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism*
D005656:Fungal Proteins