“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 |
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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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| _version_ | 1850152148690534400 |
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| author | David R.H. Evans Kendra A. Swirsding Bruce E. Taillon Jan F. Simons |
| author_facet | David R.H. Evans Kendra A. Swirsding Bruce E. Taillon Jan F. Simons |
| author_sort | David R.H. Evans |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-8ec60d2672c44f0ebddbc61234df8dff |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2004-11-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-8ec60d2672c44f0ebddbc61234df8dff2025-08-20T02:26:03ZengTaylor & Francis GroupBioTechniques0736-62051940-98182004-11-0137584084310.2144/04375PT02“One plate/three-reporter” assay format for the detection and validation of yeast two-hybrid interactionsDavid R.H. Evans0Kendra A. Swirsding1Bruce E. Taillon2Jan F. Simons31CuraGen Corporation, Branford, CT, USA1CuraGen Corporation, Branford, CT, USA1CuraGen Corporation, Branford, CT, USA1CuraGen Corporation, Branford, CT, USAWe 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.https://www.future-science.com/doi/10.2144/04375PT02 |
| spellingShingle | David R.H. Evans Kendra A. Swirsding Bruce E. Taillon Jan F. Simons “One plate/three-reporter” assay format for the detection and validation of yeast two-hybrid interactions BioTechniques |
| title | “One plate/three-reporter” assay format for the detection and validation of yeast two-hybrid interactions |
| title_full | “One plate/three-reporter” assay format for the detection and validation of yeast two-hybrid interactions |
| title_fullStr | “One plate/three-reporter” assay format for the detection and validation of yeast two-hybrid interactions |
| title_full_unstemmed | “One plate/three-reporter” assay format for the detection and validation of yeast two-hybrid interactions |
| title_short | “One plate/three-reporter” assay format for the detection and validation of yeast two-hybrid interactions |
| title_sort | one plate three reporter assay format for the detection and validation of yeast two hybrid interactions |
| url | https://www.future-science.com/doi/10.2144/04375PT02 |
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