Our protein-protein interaction assay is described in this site. We applied the split Renilla luciferase method that has been used in mammalians (Paulmurugan and Gambhir, 2003) to increase the dynamic range of PPI detection levels.
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Fig. the split Renilla luciferase |
To make a large-scale analysis possible, we established a method for Arabidopsis protoplast transfection in 96-well plates (Large-format gene transfectins). The Renilla luciferase activities are measured with a multi-well luminometer (Protein interaction). Subcellular localizations of the bait protein in the protoplasts can be analyzed by a fluorescence microscope after adding FlAsH (Protein localization). Protein amount s can be estimated by western blotting with an anti-6xhistidine (6xHis) antibody or an anti-Renilla-luciferase antibody (Protein qunatifications).
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Fig. Overview of the systems developed. |
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Reference
- N Kato, Y Fujikawa, T Fuselier, R Adamou-Dodo, A Nishitani, and MH Sato
Luminescence detection of SNARE-SNARE interaction in Arabidopsis protoplasts
Plant Molecular Biology, 2010 72:433-444 - N. Kato and J. Jones
Split luciferase complementation assay
Methods in Plant Development, A series in Methods in Molecular Biology (Ed by Lars Henning), Human Press Inc. 2010 - Y Fujikawa and N Kato
Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts.
Plant Journal, 2007 52: 185-195