title: Nanoluciferase signal brightness using furimazine substrates opens bioluminescence resonance energy transfer to widefield microscopy.
abstract: 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. © 2016 International Society for Advancement of Cytometry.
authors.affiliation: Technology Development Platform, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea.
authors.forename: Jiho
authors.initials: J
authors.lastname: Kim
authors.affiliation: Technology Development Platform, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea.
authors.forename: Regis
authors.initials: R
authors.lastname: Grailhe
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry; D008853:Microscopy; D036103:Nanotechnology; D060066:Protein Interaction Maps / Q000235:genetics; D000072105:Superoxide Dismutase-1 / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C000606290:SOD1 protein, human; C054562:yellow fluorescent protein, Bacteria; D008156:Luciferases; D000072105:Superoxide Dismutase-1
keywords: BRET; bioluminescence; fluorescence; microscopy