The Split Luciferase Complementation Assay
The Split Luciferase Complementation Assay
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DOI：
10.1007/978-1-60761-765-5_24
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发表时间：
2010-01-01
期刊：
影响因子：
--
通讯作者：
Jones, Jason
中科院分区：
文献类型：
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作者：
Kato, Naohiro;Jones, Jason
本方案中描述了一种用于研究拟南芥原生质体中蛋白质-蛋白质相互作用的裂解荧光素酶互补测定法。两个感兴趣的蛋白质，诱饵和猎物，这是基因融合的氨基和羧基端片段的海肾荧光素酶，瞬时表达在原生质体中。这些诱饵和猎物蛋白的物理相互作用重建了一些荧光素酶活性，并导致在荧光素酶底物存在下发光。然后通过微孔板光度计测量这种发光。原生质体中积累的诱饵蛋白的量可以通过使用识别肾荧光素酶的氨基末端片段的抗体的蛋白质印迹来估计。该测定的最有利方面是其以大规模形式检测感兴趣的蛋白质对的缔合和解离的能力。
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 Renal 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.