Nanoluciferase Signal Brightness Using Furimazine Substrates Opens Bioluminescence Resonance Energy Transfer to Widefield Microscopy

被引:16
作者
Kim, Jiho [1 ]
Grailhe, Regis [1 ]
机构
[1] Inst Pasteur Korea, Technol Dev Platform, 16 Daewangpangyo Ro 712 Beon Gil, Songnam 13488, Gyeonggi Do, South Korea
基金
新加坡国家研究基金会;
关键词
bioluminescence; microscopy; BRET; fluorescence;
D O I
10.1002/cyto.a.22870
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Fluorescence and bioluminescence resonance energy transfer (FRET, BRET) techniques are powerful tools for studying protein-protein interactions in cellular assays. In contrast to fluorescent proteins, chemiluminescent proteins do not require excitation light, known to trigger autofluorescence, phototoxicity, and photobleaching. Regrettably, low signal intensity of luciferase systems restricts their usage as they require specialized microscopes equipped with ultra low-light imaging cameras. In this study, we report that bioluminescence quantification in living cells using a standard widefield automated microscope dedicated to screening and high content analysis is possible with the newer luciferase systems, Nanoluciferase (Nluc). With such equipment, we showed that robust intramolecular BRET can be measured using a combination of Nluc and yellow fluorescent protein (YFP). Using the human Superoxide Dismutase 1 (SOD1) dimer model, we next validated that intermolecular BRET could be quantified at a single cell level. The enhanced signal brightness of Nluc enabling BRET imaging to widefield microscopy shows strong potential to open up single cell protein-protein interactions studies to a wider audience. (C) 2016 International Society for Advancement of Cytometry
引用
收藏
页码:742 / 746
页数:5
相关论文
共 15 条
[11]   Circumventing Photodamage in Live-Cell Microscopy [J].
Magidson, Valentin ;
Khodjakov, Alexey .
DIGITAL MICROSCOPY, 4TH EDITION, 2013, 114 :545-560
[12]  
Mo X-L, 2015, J MOL CELL BIOL
[13]   Target engagement and drug residence time can be observed in living cells with BRET [J].
Robers, Matthew B. ;
Dart, Melanie L. ;
Woodroofe, Carolyn C. ;
Zimprich, Chad A. ;
Kirkland, Thomas A. ;
Machleidt, Thomas ;
Kupcho, Kevin R. ;
Levin, Sergiy ;
Hartnett, James R. ;
Zimmerman, Kristopher ;
Niles, Andrew L. ;
Ohana, Rachel Friedman ;
Daniels, Danette L. ;
Slater, Michael ;
Wood, Monika G. ;
Cong, Mei ;
Cheng, Yi-Qiang ;
Wood, Keith V. .
NATURE COMMUNICATIONS, 2015, 6
[14]   Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis [J].
Schaub, Franz X. ;
Reza, Md. Shamim ;
Flaveny, Colin A. ;
Li, Weimin ;
Musicant, Adele M. ;
Hoxha, Sany ;
Guo, Min ;
Cleveland, John L. ;
Amelio, Antonio L. .
CANCER RESEARCH, 2015, 75 (23) :5023-5033
[15]  
Vogel Steven S, 2006, Sci STKE, V2006, pre2