“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 measuri...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2004-11-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/04375PT02 |
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| Summary: | 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. |
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| ISSN: | 0736-6205 1940-9818 |