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。
相似文献
1
Bioluminescence resonance energy transfer to detect protein-protein interactions in live cells.
Methods Mol Biol. 2015;1278:457-65. doi: 10.1007/978-1-4939-2425-7_30.
2
BRET: NanoLuc-Based Bioluminescence Resonance Energy Transfer Platform to Monitor Protein-Protein Interactions in Live Cells.
Methods Mol Biol. 2016;1439:263-71. doi: 10.1007/978-1-4939-3673-1_17.
3
Use of BRET to Study Protein-Protein Interactions In Vitro and In Vivo.
Methods Mol Biol. 2016;1443:57-78. doi: 10.1007/978-1-4939-3724-0_5.
4
Assembly and function of the regulator of G protein signaling 14 (RGS14)·H-Ras signaling complex in live cells are regulated by Gαi1 and Gαi-linked G protein-coupled receptors.
J Biol Chem. 2013 Feb 1;288(5):3620-31. doi: 10.1074/jbc.M112.440057. Epub 2012 Dec 17.
5
Bioluminescence resonance energy transfer-based imaging of protein-protein interactions in living cells.
Nat Protoc. 2019 Apr;14(4):1084-1107. doi: 10.1038/s41596-019-0129-7. Epub 2019 Mar 25.
6
Effect of enhanced Renilla luciferase and fluorescent protein variants on the Förster distance of Bioluminescence resonance energy transfer (BRET).
Biochem Biophys Res Commun. 2012 Aug 31;425(3):625-9. doi: 10.1016/j.bbrc.2012.07.133. Epub 2012 Aug 1.
7
Measuring NLR Oligomerization III: Detection of NLRP3 Complex by Bioluminescence Resonance Energy Transfer.
Methods Mol Biol. 2016;1417:159-68. doi: 10.1007/978-1-4939-3566-6_10.
8
Measuring NLR Oligomerization III: Detection of NLRP3 and NLRC4 Complex by Bioluminescence Resonance Energy Transfer.
Methods Mol Biol. 2023;2696:93-103. doi: 10.1007/978-1-0716-3350-2_6.
9
Reporter-Based BRET Sensors for Measuring Biological Functions In Vivo.
Methods Mol Biol. 2018;1790:51-74. doi: 10.1007/978-1-4939-7860-1_5.
10
Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer.
FASEB J. 2005 Dec;19(14):2017-9. doi: 10.1096/fj.05-4628fje. Epub 2005 Oct 4.
引用本文的文献
1
Signal Transduction Strategies for DNAzyme-Based Sensing and Imaging of Metal Ions in Cells and .
Chem Biomed Imaging. 2025 Mar 20;3(8):473-498. doi: 10.1021/cbmi.4c00090. eCollection 2025 Aug 25.
2
JMJD8 Regulates Adipocyte Hypertrophy Through the Interaction With Perilipin 2.
Diabetes. 2025 Apr 1;74(4):458-471. doi: 10.2337/db23-0883.
3
Exploiting Cell-Based Assays to Accelerate Drug Development for G Protein-Coupled Receptors.
Int J Mol Sci. 2024 May 17;25(10):5474. doi: 10.3390/ijms25105474.
4
Ratiometric Detection of Zn Using DNAzyme-Based Bioluminescence Resonance Energy Transfer Sensors.
Chemistry (Basel). 2023 Sep;5(3):1745-1759. doi: 10.3390/chemistry5030119. Epub 2023 Aug 8.
5
Genetically Encoded ATP Biosensors for Direct Monitoring of Cellular ATP Dynamics.
Cells. 2022 Jun 14;11(12):1920. doi: 10.3390/cells11121920.
6
Optimization of BRET saturation assays for robust and sensitive cytosolic protein-protein interaction studies.
Sci Rep. 2022 Jun 15;12(1):9987. doi: 10.1038/s41598-022-12851-9.
7
Genetically encoded fluorescent tools: Shining a little light on ER-to-Golgi transport.
Free Radic Biol Med. 2022 Apr;183:14-24. doi: 10.1016/j.freeradbiomed.2022.03.004. Epub 2022 Mar 8.
8
Molecular insights into mechanisms of GPCR hijacking by .
Proc Natl Acad Sci U S A. 2021 Oct 19;118(42). doi: 10.1073/pnas.2108856118.
9
Differential Involvement of ACKR3 C-Tail in β-Arrestin Recruitment, Trafficking and Internalization.
Cells. 2021 Mar 11;10(3):618. doi: 10.3390/cells10030618.
10
Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons.
J Biol Chem. 2021 Jan-Jun;296:100024. doi: 10.1074/jbc.RA120.016009. Epub 2020 Nov 22.
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.
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.
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.
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.
J Biol Chem. 2011 Nov 4;286(44):38659-38669. doi: 10.1074/jbc.M111.274928. Epub 2011 Aug 31.
5
Agonist-induced formation of FGFR1 homodimers and signaling differ among members of the FGF family.
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.
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.
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.
Biotechnol J. 2008 Mar;3(3):311-24. doi: 10.1002/biot.200700222.
9
Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.
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.
Proc Natl Acad Sci U S A. 2006 Jan 3;103(1):212-7. doi: 10.1073/pnas.0509763102. Epub 2005 Dec 21.
Zotero 插件