Kato Naohiro, Jones Jason
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA.
Methods Mol Biol. 2010;655:359-76. doi: 10.1007/978-1-60761-765-5_24.
A split luciferase complementation assay to study protein-protein interactions within Arabidopsis protoplasts in 96-well plates is described in this protocol. Two proteins of interest, a bait and prey, which are genetically fused to amino- and carboxy-terminal fragments of Renilla luciferase, are transiently expressed in protoplasts. Physical interactions of these bait and prey proteins reconstitute some of the luciferase activity and result in light emission in the presence of the luciferase substrate. This luminescence is then measured by a microplate luminometer. Amounts of the bait protein accumulated in the protoplasts can be estimated by Western blotting using an antibody that recognizes the amino-terminal fragment of Renilla luciferase. The most advantageous aspect of this assay is its capacity of detecting both association and dissociation of a protein pair of interest in a large-scale format.
本方案介绍了一种用于在96孔板中研究拟南芥原生质体内蛋白质-蛋白质相互作用的分裂荧光素酶互补分析方法。两种感兴趣的蛋白质，即诱饵蛋白和猎物蛋白，分别与海肾荧光素酶的氨基末端和羧基末端片段进行基因融合，并在原生质体中瞬时表达。这些诱饵蛋白和猎物蛋白的物理相互作用会重建部分荧光素酶活性，并在荧光素酶底物存在的情况下导致发光。然后用微孔板发光计测量这种发光。原生质体中积累的诱饵蛋白量可以通过使用识别海肾荧光素酶氨基末端片段的抗体进行蛋白质印迹来估计。该分析方法最有利的方面是其能够大规模检测感兴趣的蛋白质对的结合和解离。
