Direct protein–protein interaction was tested by Y2H technique according to the yeast protocols handbook and the Matchmaker GAL4 Two-hybrid System 3 manual (both Clontech, Heidelberg, Germany). Yeast strain Y190 was cotransformed with respective plasmids, followed by the selection of transformants on medium lacking Leu and Trp at 30 °C for 3 d and the subsequent transfer to medium lacking Leu, Trp, and His (supplemented with 25 mm 3-amino-triazole) for growth selection. Cells growing on selective medium were further tested for activity of the lacZ reporter gene using filter lift assays.
Gal4 two hybrid system 3 manual
Manufactured by Takara Bio
The GAL4 Two-hybrid System 3 manual provides detailed instructions for the GAL4 Two-hybrid System 3, a method used to study protein-protein interactions. The manual outlines the core function and technical specifications of the system.
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Yeast Two-Hybrid Protein Interaction Assay
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Direct Protein-Protein Interaction via Y2H
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Direct protein-protein interaction was tested by Y2H technique according to the yeast protocols handbook and the Matchmaker GAL4 Two-hybrid System 3 manual (both Clontech, Heidelberg, Germany). Yeast strain Y190 was co-transformed with respective plasmids, followed by selection of transformants on medium lacking Leu and Trp at 30°C for 3 days and subsequent transfer to medium lacking Leu, Trp and His (supplemented with 25 mM 3-aminotriazole) for growth selection. Cells growing on selective medium were further tested for activity of the lacZ reporter gene using filter lift assays.
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