Improved yellow-green split fluorescent proteins for protein labeling and signal amplification
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Abstract:
The flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP11 tags. To improve the FP1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A1-10/11 and mNG3K1-10/11) and one mClover-based split FP (CloGFP1-10/11). mNG3A1-10/11 and mNG3K1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG211 fragments in mammalian cells.Keywords:
Fluorescent labelling
Inspired by the green fluorescent protein (GFP), a novel amphiphilic fluorescent polymer was designed and synthesized. The amphiphilic polymer showed enhanced fluorescent properties after self-assembly into micellar aggregates. Moreover, the fluorescence enhancement behavior of the GFP-inspired polymer through self-assembly was successfully applied in cell imaging.
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The fluorescence of green fluorescent protein (GFP), present both within whole yeast cells and in protein extracts from yeast cells, has been observed to be significantly polarised. Fluorescence polarisation is proposed as a useful technique to allow some discrimination between GFP fluorescence and that of other interfering species in cell or media matrices, which have fluorescence bands that overlap those of GFP, which should lead to improved resolution and limits of detection. The method has been tested by discriminating between the fluorescence of GFP in cell extracts and added fluorescein, both of which fluoresce brightly at the same wavelength. The flow-through instrumentation incorporating an argon-ion laser developed for this work is also described.
Bimolecular fluorescence complementation
Fluorescent protein
Fluorescent labelling
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Fluorescent labelling
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The jellyfish green fluorescent protein (GFP) is a versatile biological marker and reporter. Here we demonstrate that marked polarisation in GFP fluorescence, upon excitation with plane polarised light, can be exploited to enhance the detection of GFP when in the presence of cellular auto-fluorescence and other fluorescent compounds that emit at the same wavelength. The development of flow-though instrumentation dedicated to the sensitive detection of GFP by fluorescence polarisation is described, and used in both a continuous flow and flow-injection format. The intensity, spectral properties and extent of polarisation in the auto-fluorescence of various yeast strains and growth media are investigated. The application of fluorescence polarisation is shown to enhance the measurement of the induction of GFP in yeast cells, genetically modified to produce GFP as an indicator of DNA damage, compared with a conventional fluorescence method.
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Fluorescent labeling of reovirus virions with succinimidyl ester-conjugated fluorescent probes
Fluorescent labelling
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Fluorescent protein
Fluorescent labelling
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Fluorescent labelling
Fluorescent protein
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