利用生物发光共振能量转移检测活细胞中的蛋白质-蛋白质相互作用。
Bioluminescence resonance energy transfer to detect protein-protein interactions in live cells.
作者信息
Brown Nicole E, Blumer Joe B, Hepler John R
机构信息
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, 30322, USA.
出版信息
Methods Mol Biol. 2015;1278:457-65. doi: 10.1007/978-1-4939-2425-7_30.
Abstract
Bioluminescence resonance energy transfer (BRET) is a valuable tool to detect protein-protein interactions. BRET utilizes bioluminescent and fluorescent protein tags with compatible emission and excitation properties, making it possible to examine resonance energy transfer when the tags are in close proximity (<10 nm) as a typical result of protein-protein interactions. Here we describe a protocol for detecting BRET from two known protein binding partners (Gαi1 and RGS14) in HEK 293 cells using Renilla luciferase and yellow fluorescent protein tags. We discuss the calculation of the acceptor/donor ratio as well as net BRET and demonstrate that BRET can be used as a platform to investigate the regulation of protein-protein interactions in live cells in real time.
摘要
生物发光共振能量转移（BRET）是检测蛋白质-蛋白质相互作用的一种有价值的工具。BRET利用具有兼容发射和激发特性的生物发光和荧光蛋白标签，当标签由于蛋白质-蛋白质相互作用而紧密接近（<10 nm）时，能够检测共振能量转移。在这里，我们描述了一种使用海肾荧光素酶和黄色荧光蛋白标签在HEK 293细胞中检测来自两个已知蛋白质结合伴侣（Gαi1和RGS14）的BRET的方案。我们讨论了受体/供体比率以及净BRET的计算，并证明BRET可作为实时研究活细胞中蛋白质-蛋白质相互作用调节的平台。
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本文引用的文献
1
Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells.建立生物发光共振能量转移高通量筛选检测法，以在哺乳动物细胞中寻找蛋白质/蛋白质相互作用抑制剂。
Front Endocrinol (Lausanne). 2012 Sep 11;3:100. doi: 10.3389/fendo.2012.00100. eCollection 2012.
2
Sensitive and high resolution localization and tracking of membrane proteins in live cells with BRET.利用 BRET 技术在活细胞中对膜蛋白进行敏感且高分辨率的定位和追踪。
Traffic. 2012 Nov;13(11):1450-6. doi: 10.1111/j.1600-0854.2012.01401.x. Epub 2012 Aug 5.
3
Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling.荧光/生物发光共振能量转移技术在 G 蛋白偶联受体激活和信号转导研究中的应用。
Pharmacol Rev. 2012 Apr;64(2):299-336. doi: 10.1124/pr.110.004309. Epub 2012 Mar 8.
4
G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14·Gαi1 complex in live cells.G 蛋白偶联受体和对乙酰胆碱酯酶抑制剂-8A（Ric-8A）的抗性都可调节活细胞中的 G 蛋白信号转导调节因子 14（RGS14）·Gαi1 复合物。
J Biol Chem. 2011 Nov 4;286(44):38659-38669. doi: 10.1074/jbc.M111.274928. Epub 2011 Aug 31.
5
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Biochem Biophys Res Commun. 2011 Jun 17;409(4):764-8. doi: 10.1016/j.bbrc.2011.05.085. Epub 2011 May 20.
6
Regulation of the AGS3·G{alpha}i signaling complex by a seven-transmembrane span receptor.七跨膜螺旋受体对 AGS3·Gαi 信号复合物的调节。
J Biol Chem. 2010 Oct 29;285(44):33949-58. doi: 10.1074/jbc.M110.138073. Epub 2010 Aug 17.
7
Receptor-regulated interaction of activator of G-protein signaling-4 and Galphai.G 蛋白信号转导激活因子 4 和 Galphai 的受体调节相互作用。
J Biol Chem. 2010 Jul 2;285(27):20588-94. doi: 10.1074/jbc.C109.088070. Epub 2010 May 7.
8
The BRET technology and its application to screening assays.生物发光共振能量转移（BRET）技术及其在筛选分析中的应用。
Biotechnol J. 2008 Mar;3(3):311-24. doi: 10.1002/biot.200700222.
9
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J Biol Chem. 2007 Apr 6;282(14):10576-84. doi: 10.1074/jbc.M609695200. Epub 2007 Feb 5.
10
Gialpha and Gbeta subunits both define selectivity of G protein activation by alpha2-adrenergic receptors.Gα和Gβ亚基均决定α2肾上腺素能受体对G蛋白激活的选择性。
Proc Natl Acad Sci U S A. 2006 Jan 3;103(1):212-7. doi: 10.1073/pnas.0509763102. Epub 2005 Dec 21.
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