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TECHNICAL ADVANCE: Split luciferase complementation assay to study protein–protein interactions in Arabidopsis protoplasts
Author(s) -
Fujikawa Yukichi,
Kato Naohiro
Publication year - 2007
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2007.03214.x
Subject(s) - luciferase , complementation , arabidopsis , protein fragment complementation assay , protoplast , biology , recombinant dna , microbiology and biotechnology , dna , biochemistry , gene , chemistry , transfection , mutant
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.