scholarly journals Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy

2017 ◽  
Author(s):  
Alessia Lepore ◽  
Hannah Taylor ◽  
Dirk Landgraf ◽  
Burak Okumus ◽  
Sebastián Jaramillo-Riveri ◽  
...  
Keyword(s):  
Cell Biology ◽  
Quantitative Methods ◽  
Fluorescent Protein ◽  
Detection Efficiency ◽  
Fluorescent Proteins ◽  
Attractive Alternative ◽  
Highly Sensitive ◽  
Endogenous Proteins ◽  
Photo Stability ◽  
Better Than

ABSTRACTCell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (<7) in individualEscherichia colicells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is ≥80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolboxThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 515-522 ◽  
Author(s):  
Preetinder K. Dhanoa ◽  
Alison M. Sinclair ◽  
Robert T. Mullen ◽  
Jaideep Mathur
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Imaging ◽  
Living Cells ◽  
Special Issue ◽  
Exciting Possibility ◽  
Selection Of

The discovery and development of multicoloured fluorescent proteins has led to the exciting possibility of observing a remarkable array of subcellular structures and dynamics in living cells. This minireview highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

scholarly journals mCherry contains a fluorescent protein isoform that interferes with its reporter function

2021 ◽  
Author(s):  
Maxime Fages-Lartaud ◽  
Lisa Tietze ◽  
Florence Elie ◽  
Rahmi Lale ◽  
Martin Frank Hohmann-Marriott
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Red Fluorescent Protein ◽  
Functional Protein ◽  
Red Fluorescent Proteins ◽  
Expression Studies ◽  
Protein Isoform

AbstractFluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

Studying the Cell Biology of Apicomplexan Parasites Using Fluorescent Proteins

2004 ◽  
Vol 10 (5) ◽  
pp. 568-579 ◽  
Author(s):  
Marc-Jan Gubbels ◽  
Boris Striepen
Keyword(s):  
Protein Trafficking ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Biological Processes ◽  
Organelle Biogenesis ◽  
Apicomplexan Parasites ◽  
Experimental Protocols ◽  
Green Fluorescent

The ability to transfect Apicomplexan parasites has revolutionized the study of this important group of pathogens. The function of specific genes can be explored by disruption of the locus or more subtly by introduction of altered or tagged versions. Using the transgenic reporter gene green fluorescent protein (GFP), cell biological processes can now be studied in living parasites and in real time. We review recent advances made using GFP-based experiments in the understanding of protein trafficking, organelle biogenesis, and cell division inToxoplasma gondiiandPlasmodium falciparum. A technical section provides a collection of basic experimental protocols for fluorescent protein expression inT. gondii. The combination of thein vivomarker GFP with an increasingly diverse genetic toolbox forT. gondiiopens many exciting experimental opportunities, and emerging applications of GFP in genetic and pharmacological screens are discussed.

scholarly journals CFP and YFP, but Not GFP, Provide Stable Fluorescent Marking of Rat Hepatic Adult Stem Cells

2008 ◽  
Vol 2008 ◽  
pp. 1-9 ◽  
Author(s):  
Rouzbeh R. Taghizadeh ◽  
James L. Sherley
Keyword(s):  
Stem Cells ◽  
Time Scales ◽  
Cell Biology ◽  
Adult Stem Cells ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Fluorescent Reporter ◽  
Green Fluorescent

The stable expression of reporter genes in adult stem cells (ASCs) has important applications in stem cell biology. The ability to integrate a noncytotoxic, fluorescent reporter gene into the genome of ASCs with the capability to track ASCs and their progeny is particularly desirable for transplantation studies. The use of fluorescent proteins has greatly aided the investigations of protein and cell function on short-time scales. In contrast, the obtainment of stably expressing cell strains with low variability in expression for studies on longer-time scales is often problematic. We show that this difficulty is partly due to the cytotoxicity of a commonly used reporter, green fluorescent protein (GFP). To avoid GFP-specific toxicity effects during attempts to stably mark a rat hepatic ASC strain and, therefore, obtain stable, long-term fluorescent ASCs, we evaluated cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), in addition to GFP. Although we were unable to derive stable GFP-expressing strains, stable fluorescent clones (up to 140 doublings) expressing either CFP or YFP were established. When fluorescently marked ASCs were induced to produce differentiated progeny cells, stable fluorescence expression was maintained. This property is essential for studies that track fluorescently marked ASCs and their differentiated progeny in transplantation studies.

scholarly journals Going Viral with Fluorescent Proteins

2015 ◽  
Vol 89 (19) ◽  
pp. 9706-9708 ◽  
Author(s):  
Lindsey M. Costantini ◽  
Erik L. Snapp
Keyword(s):  
Physical Properties ◽  
Fluorescence Imaging ◽  
Viral Protein ◽  
Cell Biology ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Imaging Techniques ◽  
Cell Interactions

Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

scholarly journals Nuclear Import Analysis of Two Different Fluorescent Marker Proteins into Hepatocyte Cell Lines (HuH-7 Cell)

2015 ◽  
Vol 10 (2) ◽  
Author(s):  
Aris Haryanto ◽  
Michael Kann
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Intracellular Localization ◽  
Living Cells ◽  
Marker Protein ◽  
Dna Encoding ◽  
Protein Fusion ◽  
Fluorescent Marker ◽  
Localization Study

The application of fluorescent proteins as expression markers and protein fusion partners has provedimmensely valuable for resolving the organization of biological events in living cells. EGFP and DsRed2 arecommonly fluorescent marker protein which is used for biotechnology and cell biology research. The presentstudy was designed to identify the expression vector that suitable to ligate with DNA encoding HBV coreprotein for intracellular localization study in hepatocyte cell, which were expressed as fusion proteins. We alsocompared and quantified the expressed fluorescent protein which predominantly localized in the cellcompartment. The results indicated that DsRed2 shown as less than ideal for intracellular localization study ofthan EGFP, because of its tetrameric structure of the fluorescent protein and when fused to a protein of interest,the fusion protein often forms aggregates in the living cells. In contrast, EGFP fluorescent protein shown a muchhigher proportion of cytoplasmic localization, thus being more suitable for analysis of intracellular localizationthan DsRed2 fluorescent protein. EGFP fluorescent protein is also capable to produce a strong green fluorescencewhen excited by blue light, without any exogenously added substrate or cofactor, events inside living cell canthus be visualized in a non-invasive way. Based on our present quantitative data and some reasons above shownthat EGFP is more suitable than DsRed2 as a fluorescent marker protein for intracellular localization study intoHuH-7 cell.Keywords: EGFP, DsRed2 fluorescent protein , HuH-7 cell, HBV, intracellular localization

scholarly journals Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

2020 ◽  
Author(s):  
Jérôme Goudeau ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D. Leonetti ◽  
Andrew G. York ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

AbstractWe create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest and can be detected wherever the large fragment is expressed and complemented. There is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet, and generate transgenic C. elegans lines to allow easy single-color labeling in muscles and dual-color labeling in somatic cells. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for an easy, cloning-free method for CRISPR/Cas9 editing.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light &gt;600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

A Three-Dimensional Tetraphenylethylene-Based Fluorescence Covalent Organic Framework for Molecular Recognition

2020 ◽  
Author(s):  
Junxia Ren ◽  
Yaozu Liu ◽  
Xin Zhu ◽  
Yangyang Pan ◽  
Yujie Wang ◽  
...  
Keyword(s):  
Molecular Recognition ◽  
Fluorescence Probe ◽  
Three Dimensional ◽  
Hazardous Substances ◽  
Covalent Organic Framework ◽  
Highly Sensitive ◽  
Volatile Organic ◽  
Polycyclic Aromatic ◽  
Better Than ◽  
Organic Framework

<p><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a>The development of highly-sensitive recognition of </a><a></a><a></a><a></a><a></a><a>hazardous </a>chemicals, such as volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs), is of significant importance because of their widespread social concerns related to environment and human health. Here, we report a three-dimensional (3D) covalent organic framework (COF, termed JUC-555) bearing tetraphenylethylene (TPE) side chains as an aggregation-induced emission (AIE) fluorescence probe for sensitive molecular recognition.<a></a><a> </a>Due to the rotational restriction of TPE rotors in highly interpenetrated framework after inclusion of dimethylformamide (DMF), JUC-555 shows impressive AIE-based strong fluorescence. Meanwhile, owing to the large pore size (11.4 Å) and suitable intermolecular distance of aligned TPE (7.2 Å) in JUC-555, the obtained material demonstrates an excellent performance in the molecular recognition of hazardous chemicals, e.g., nitroaromatic explosives, PAHs, and even thiophene compounds, via a fluorescent quenching mechanism. The quenching constant (<i>K</i><sub>SV</sub>) is two orders of magnitude better than those of other fluorescence-based porous materials reported to date. This research thus opens 3D functionalized COFs as a promising identification tool for environmentally hazardous substances.</p>

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