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TECHNICAL ADVANCE: Split luciferase complementation assay to study protein–protein interactions in Arabidopsis protoplasts
Article first published online: 30 JUL 2007
DOI: 10.1111/j.1365-313X.2007.03214.x
Additional Information
How to Cite
Fujikawa, Y. and Kato, N. (2007), TECHNICAL ADVANCE: Split luciferase complementation assay to study protein–protein interactions in Arabidopsis protoplasts. The Plant Journal, 52: 185–195. doi: 10.1111/j.1365-313X.2007.03214.x
Publication History
- Issue published online: 30 JUL 2007
- Article first published online: 30 JUL 2007
- Received 21 February 2007; revised 29 May 2007; accepted 5 June 2007.
Keywords:
- protein–protein interactions;
- split luciferase;
- in vivo;
- protoplasts;
- Arabidopsis;
- FlAsH
Summary
We developed a split luciferase complementation assay to study protein–protein interactions in Arabidopsis protoplasts. In this assay, the N- and C-terminal fragments of Renilla reniforms luciferase are translationally fused to bait and prey proteins, respectively. When the proteins interact, split luciferase becomes activated and emits luminescence that can be measured by a microplate luminometer. Split luciferase activity was measured by first transforming protoplasts with a DNA vector in a 96-well plate. DNA vector expressing both bait and prey genes was constructed through two independent in vitro DNA recombinant reactions, Gateway and Cre-loxP. As proof of concept, we detected the protein–protein interactions between the nuclear histones 2A and 2B, as well as between membrane proteins SYP (syntaxin of plant) 51 and SYP61, in Arabidopsis protoplasts.