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Initially, we were faced with the decision of how to efficiently construct and screen libraries of DsRed mutants such that we could direct the evolution of tetrameric DsRed toward a mRFP. Ultimately the best solution was to take the semirational approach of breaking the dimer interfaces in a stepwise fashion (first AB then AC) and undertaking a directed evolution strategy to rescue the red fluorescence. This combination of targeted and random mutagenesis successfully directed the evolution of DsRed from the poorly fluorescent dimer T1-I125R to the monomeric mRFP1 in eight generations. In retrospect, breaking up the tetramer was not the barrier to the discovery of mRFP1; the challenge was to find the correct combination of many mutations to rescue the red fluorescence in the crippled dimers and monomers.. There are likely several mechanisms by which mutations in dimer2 and mRFP1 have contributed to rescuing the red fluorescence. Although one mechanism probably involves improving the folding ...
Initially, we were faced with the decision of how to efficiently construct and screen libraries of DsRed mutants such that we could direct the evolution of tetrameric DsRed toward a mRFP. Ultimately the best solution was to take the semirational approach of breaking the dimer interfaces in a stepwise fashion (first AB then AC) and undertaking a directed evolution strategy to rescue the red fluorescence. This combination of targeted and random mutagenesis successfully directed the evolution of DsRed from the poorly fluorescent dimer T1-I125R to the monomeric mRFP1 in eight generations. In retrospect, breaking up the tetramer was not the barrier to the discovery of mRFP1; the challenge was to find the correct combination of many mutations to rescue the red fluorescence in the crippled dimers and monomers.. There are likely several mechanisms by which mutations in dimer2 and mRFP1 have contributed to rescuing the red fluorescence. Although one mechanism probably involves improving the folding ...
Green-to-red photoconversion is a reaction that occurs in a limited number of fluorescent proteins and that is currently mechanistically debated. In this contribution, we report on our investigation of the photoconvertible fluorescent protein Dendra2 by employing a combination of pump-probe, up-conversion and single photon timing spectroscopic techniques. Our findings indicate that upon excitation of the neutral green state an excited state proton transfer proceeds with a time constant of 3.4 ps between the neutral green and the anionic green states. In concentrated solution we detected resonance energy transfer (25 ps time constant) between green and red monomers. The time-resolved emission spectra suggest also the formation of a super-red species, first observed for DsRed (a red fluorescent protein from the corallimorph species Discosoma) and consistent with peculiar structural details present in both proteins.
Author(s): Shaner, Nathan Christopher | Abstract: Fluorescent proteins are intrinsically fluorescent, genetically encodable tags that can be expressed in many heterologous organisms. Originally cloned from jellyfish and corals, these proteins and their engineered derivatives have become ubiquitous tools in molecular and cell biology. While wild-type fluorescent proteins sometimes possess sufficiently beneficial properties to be used unmodified, many applications require improvements in brightness or photostability, reduction of oligomerization, or other specific properties that require additional engineering of the wild-type protein. This dissertation presents experiments drawn from the entire spectrum of fluorescent protein science, from the cloning of novel wild-type fluorescent proteins to the engineering of wavelength-shifted, photostable, and photoactivatable variants of existing fluorescent proteins. The previously engineered monomeric red fluorescent protein, mRFP1, was engineered through a
Read pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants, Plant Molecular Biology on DeepDyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips.
mKate2 is the next generation of monomeric far-red fluorescent protein TagFP635 (mKate) [Shcherbo et al., 2007; Shcherbo et al., 2009]. Possessing fluorescence with excitation/emission maxima at 588 and 633 nm, mKate2 is almost 3-fold brighter than TagFP635 and is 10-fold brighter than mPlum at the physiological pH = 7.5. Within the optical window optimal for light penetration in living tissues, calculated brightness of mKate2 is at least 2-fold higher compared to any monomeric fluorescent protein reported to date. mKate2 is characterized by complete and fast chromophore maturation at 37°C with maturation half-time ,20 min (versus 40 min for mCherry). It is more photostable under both widefield and confocal illumination than other monomeric far-red proteins, including TagFP635, mRaspberry and mPlum. The high brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior ...
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Supplementary MaterialsSupplementary Details Supplementary information srep00688-s1. results in the conformational dynamics from the RFP chromophore. The genetically-encoded fluorescent proteins (FPs) are effective equipment for imaging in biology1. The prototypical green fluorescent proteins (GFP) in the jellyfish includes an 11-stranded -barrel encircling the 4-(p-hydroxybenzylidene)imidazolidin-5-one chromophore, which is normally produced autocatalytically1. The crimson fluorescent proteins (DsRed) from coral isomerization from the chromophore in the thrilled state, regarding rotation around imidazolinone exocyclic connection (I-bond)6. Furthermore to I-bond isomerization, the rotation around phenyl (P-) connection may also be extremely efficient (occasionally barrierless and on picosecond timescale) in the isolated chromophore7,8,9,10,11,12,13,14. In mFruits and DsRed, fast reversible bleaching seen in mass tests15,16, fluorescence relationship17,18,19,20, and one molecule spectroscopy20,21 ...
Simple and easy to engineer metal-sensing molecules that are capable of differentiating metal ions and producing metal-specific signals are highly desirable. Metal ions affect the thermal stability of proteins by increasing or decreasing their resistance to unfolding. This work illustrates a new strategy for designing bivalent fluorescent fusion proteins capable of differentiating metal ions in solution through their distinct effects on a proteins thermal stability. A new dual purpose metal sensor was developed consisting of biotin protein ligase (BirA) from B. pseudomallei (Bp) fused to green fluorescent protein (GFP). When coupled with differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) for signal-transduction detection, Bp BirA-GFP yields distinct protein unfolding signatures with Zn(II) and Cu(II) ions in aqueous solutions. The limit of detection of the system is ∼1 μM for both metal species. The system can be used in a variety of high-throughput assay formats including ...
Plasmid AAV-FLEX-rev-ChR2-tdtomato from Dr. Scott Sternsons lab contains the insert Channelrhodopsin 2-tdtomato and is published in J Neurosci. 2008 Jul 9. 28(28):7025-30. This plasmid is available through Addgene.
Plasmid 20XUAS IVS CsChrimson tdtomato_tr from Dr. Vivek Jayaramans lab contains the insert Chrimson_tdtomato_trafficked and is published in Nat Methods. 2014 Mar;11(3):338-46. Epub 2014 Feb 9. This plasmid is available through Addgene.
This X-linked targeted knock-in strain co-marks cells expressing the Foxp3 (forkhead box P3) gene with monomeric red fluorescent protein (mRFP). RFP expression faithfully marks gene expression in lymphocytes. This strain may be helpful in studies of Foxp3-expressing regulatory T cells.
Author: Reiländer, Helmut et al.; Genre: Journal Article; Published in Print: 1996-02-06; Title: Functional Expression of the Aequorea victoria Green Fluorescent Protein in Insect Cells Using the Baculovirus Expression System
Structural investigations on green fluorescent protein variants and the adenylyl cyclase associated protein [Elektronische Ressource] / Dorota Ksiazek : Technische Universität München Institut für Organische Chemie und Biochemie Max-Planck-Institut für Biochemie Abteilung Strukturforschung Biologische NMR-Arbeitsgruppe Structural Investigations on Green Fluorescent Protein Variants and the Adenylyl Cyclase-Associated Protein Dorota Ksiazek Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur
TY - JOUR. T1 - Green fluorescent protein mutant as label in homogeneous assays for biomolecules. AU - Deo, Sapna K.. AU - Daunert, Sylvia. PY - 2001/2/1. Y1 - 2001/2/1. N2 - The green fluorescent protein (GFP) and its mutants have been extensively used to study various cellular processes and, more recently, as labels in binding assays. We have employed a mutant of GFP, an enhanced GFP (EGFP), in the development of homogeneous assays for biotin and cortisol. To demonstrate the feasibility of using EGFP as a label with different kinds of binders in the development of homogeneous assays, we employed the biotin-avidin and an antigen-antibody as the binding pairs. Biotin and cortisol were chemically conjugated to EGFP. A quenching of fluorescence intensity of EGFP was observed upon binding of avidin to the EGFP-biotin conjugate. The percentage fluorescence quenching observed decreased as the concentration of free biotin in the sample increased due to the fewer binding sites on avidin available for ...
As a member of the wwPDB, the RCSB PDB curates and annotates PDB data according to agreed upon standards. The RCSB PDB also provides a variety of tools and resources. Users can perform simple and advanced searches based on annotations relating to sequence, structure and function. These molecules are visualized, downloaded, and analyzed by users who range from students to specialized scientists.
TY - JOUR. T1 - Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large stokes shift. AU - Piatkevich, Kiryl D.. AU - Malashkevich, Vladimir N.. AU - Almo, Steven C.. AU - Verkhusha, Vladislav. PY - 2010/8/11. Y1 - 2010/8/11. N2 - LSSmKate1 and LSSmKate2 are monomeric red fluorescent proteins (RFPs) with large Stokes shifts (LSSs), which allows for efficient separation of absorbance and emission maxima, as well as for excitation with conventional two-photon laser sources. These LSSmKates differ by a single amino acid substitution at position 160 and exhibit absorbance maxima around 460 nm, corresponding to a neutral DsRed-like chromophore. However, excitation at 460 nm leads to fluorescence emission above 600 nm. Structures of LSSmKate1 and LSSmKate2, determined at resolutions of 2.0 and 1.5, respectively, revealed that the predominant DsRed-chromophore configurations are cis for LSSmKate1 but trans for LSSmKate2. Crystallographic and mutagenesis ...
TY - JOUR. T1 - FMDV replicons encoding green fluorescent protein are replication competent. AU - Tulloch, Fiona. AU - Pathania, Uday. AU - Luke, Garry A.. AU - Nicholson, John. AU - Stonehouse, Nicola J.. AU - Rowlands, David J.. AU - Jackson, Terry. AU - Tuthill, Toby. AU - Haas, Juergen. AU - Lamond, Angus I.. AU - Ryan, Martin D.. PY - 2014/12/1. Y1 - 2014/12/1. N2 - The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious replicon systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional (L pro) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs ...
Supplementary Materialsoncotarget-09-6518-s001. This demonstrates the significant potential of alveolar type II cells in orchestrating the process of metastasis, rendering it as one of the target cell types of the lung of therapeutic importance in human NSCLC. expression is usually replaced with reddish fluorescent protein (DsRed*MST) expression in tissues expressing Cre recombinase. We utilized transgenic mice in which the human SPC (Sftpc) gene promoter is used expressing the invert tetracycline transactivator (rtTA) hence placing the appearance of Cre-recombinase (CRE) beneath the conditional control of doxycycline. Appearance of Cre was utilized to completely label cells with Crimson fluorescent proteins (DsRed) in alveolar type II cells. Distinctive lines of transgenic mice that exhibit rtTA beneath the control of the individual surfactant-associated proteins C (Sftpc/SPC) gene promoter had been bred VE-821 enzyme inhibitor to TetO-Cre mice and reporter mice (LacZ/DsRed) creating triple ...
Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0- and 1.9-A resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes. roGFP1 has been ...
Aequoria victoria green fluorescent protein (GFP) is a revolutionary molecular biology tool because of its spontaneous peptide backbone cyclization and chromophore formation from residues Ser65, Tyr66, and Gly67. Here we use structure-based design, comprehensive targeted mutagenesis, and high-resolution crystallography to probe the significant functional role of conserved Arg96 (R96) in chromophore maturation. The R96M GFP variant, in which the R96M side chain is similar in volume but lacks the R96 positive charge, exhibits dramatically slower chromophore maturation kinetics (from hours to months). Comparison of the precyclized conformation of the chromophore-forming residues with the mature R96M chromophore reveals a similar Y66 conformer, contrary to the large Y66 conformational change previously defined in the slowly maturing R96A variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116]. Comprehensive ...
References for The Development of New FPs 15. Shaner , N C, Patterson, G H, & Davidson, M W. (2007). Advances in fluorescent protein technology . Journal of Cell Science, 120. PMID: 18057027 16. Shaner, N C, Steinbach, P A & Tsien, R Y. (2005). A guide to choosing fluorescent proteins. Nature Methods, 2, PMID: 16299475 17. Ai, H, Shaner, N C, Cheng, Z, Tsien, R Y & Campbell, R E. (2007). Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry, 46, PMID: 17444659 18. Kremers, G J, Goedhart, J, van den Heuvel, D J, Gerritsen, H C. & Gadella, T W J. (2007). Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells. Biochemistry, 46, PMID: 17323929 19. Cubitt, A B., Heim, R, Adams, S R, Boyd, A E, Gross, L A & Tsien, R Y. (1995). Understanding, improving and using green fluorescent proteins. Trends Biochemical Science, 20, PMID: 8578587 20. Rizzo, M A, Springer, G H, Granada, B & Piston, D W. (2004). ...
References for The Development of New FPs 15. Shaner , N C, Patterson, G H, & Davidson, M W. (2007). Advances in fluorescent protein technology . Journal of Cell Science, 120. PMID: 18057027 16. Shaner, N C, Steinbach, P A & Tsien, R Y. (2005). A guide to choosing fluorescent proteins. Nature Methods, 2, PMID: 16299475 17. Ai, H, Shaner, N C, Cheng, Z, Tsien, R Y & Campbell, R E. (2007). Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry, 46, PMID: 17444659 18. Kremers, G J, Goedhart, J, van den Heuvel, D J, Gerritsen, H C. & Gadella, T W J. (2007). Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells. Biochemistry, 46, PMID: 17323929 19. Cubitt, A B., Heim, R, Adams, S R, Boyd, A E, Gross, L A & Tsien, R Y. (1995). Understanding, improving and using green fluorescent proteins. Trends Biochemical Science, 20, PMID: 8578587 20. Rizzo, M A, Springer, G H, Granada, B & Piston, D W. (2004). ...
As a member of the wwPDB, the RCSB PDB curates and annotates PDB data according to agreed upon standards. The RCSB PDB also provides a variety of tools and resources. Users can perform simple and advanced searches based on annotations relating to sequence, structure and function. These molecules are visualized, downloaded, and analyzed by users who range from students to specialized scientists.
We present recent data on dynamic imaging of Rac1 activity in live T-cells. Förster resonance energy transfer between enhanced green and monomeric red fluorescent protein pairs which form part of a biosensor molecule provides a metric of this activity. Microscopy is performed using a multi-functional high-content screening instrument using fluorescence anisotropy to provide a means of monitoring protein-protein activity with high temporal resolution. Specifically, the response of T-cells upon interaction of a cell surface receptor with an antibody coated multi-well chamber was measured. We observed dynamic changes in the activity of the biosensor molecules with a time resolution that is difficult to achieve with traditional methodologies for observing Förster resonance energy transfer (fluorescence lifetime imaging using single photon counting or frequency domain techniques) and without spectral corrections that are normally required for intensity based methodologies.
Intensive searches for novel green fluorescent protein (GFP)-like fluorescent proteins have identified more than 150 distinct genes that, together with its mutants, cover the excitation range from 380 to 600 nanometers (nm) and the emission range from 440 to 650 nm (see table below). Despite spectral diversity, a family of GFP-like proteins possesses common significant structural, biochemical and photophysical features. Many of these spectroscopically active proteins are developed to commercially available genetically-encoded fluorescent probes. In comparison to other natural pigments and fluorophores, GFP-like proteins stand out because they form internal chromophores without requiring accessory cofactors, external enzymatic catalysis or substrates other than molecular oxygen. It gives GFP-like proteins many advantages including that chromophore formation is possible in live organisms, tissues or cells while maintaining their integrity as well as molecular, organelle and tissue targeting and ...
The recent emergence of an autofluorescent protein, the green fluorescent protein (GFP), has opened the door for the convenient use of intact living cells and organisms as experimental systems in fields ranging from cell biology to biomedicine. We present an overview of some of the major applications of GFP, namely its use in protein tagging and in monitoring gene expression as well as its potential in a variety of biological screens.. ...
Fluorescent proteins are likely one of the most famous research tools derived from bioprospecting. Examples include dsRed as well as GFP and its many derivatives, which have been utilized throughout biological research. Interestingly, these fluorescent proteins are finding new purpose in medicine as visual guides during surgery. Before tumorectomy, a mouse with internal tumors is injected with a recombinant form of GFP, which is targeted to and accumulates on the cells of blood vessels. During surgical removal of the tumor, the introduced GFP provides a surgeon with a strong visual queue of nearby blood vessels greatly reducing the risk of blood vessel lacerations. DNA and RNA polymerases are the workhorses of modern biotechnology. Almost every aspect of modern biological research depends upon nucleic acid polymerases in one way or another. Recombinant cloning techniques, Sanger sequencing, and qPCR cover a few of the most common uses. These examples also highlight the shared importance of ...
TY - JOUR. T1 - DNA sequence-enabled reassembly of the green fluorescent protein. AU - Stains, Cliff I.. AU - Porter, Jason R.. AU - Ooi, Aik T.. AU - Segal, David J.. AU - Ghosh, Indraneel. PY - 2005/8/10. Y1 - 2005/8/10. N2 - We describe a general methodology for the direct detection of DNA by the design of a split-protein system that reassembles to form an active complex only in the presence of a targeted DNA sequence. This approach, called SEquence Enabled Reassembly (SEER) of proteins, combines the ability to rationally dissect proteins to construct oligomerization-dependent protein reassembly systems and the availability of DNA binding Cys2-His2 zinc-finger motifs for the recognition of specific DNA sequences. We demonstrate the feasibility of the SEER approach utilizing the split green fluorescent protein appended to appropriate zinc fingers, such that chromophore formation is only catalyzed in the presence of DNA sequences that incorporate binding sites for both zinc fingers.. AB - We ...
TY - JOUR. T1 - Structural characterization of the photoswitchable fluorescent protein Dronpa-C62S. AU - Nam, Ki Hyun. AU - Kwon, Oh Yeun. AU - Sugiyama, Kanako. AU - Lee, Won Ho. AU - Kim, Young Kwan. AU - Song, Hyun Kyu. AU - Kim, Eunice Eunkyung. AU - Park, Sam Yong. AU - Jeon, Hyesung. AU - Hwang, Kwang Yeon. N1 - Funding Information: We thank Dr. H.S. Lee and his staff for assistance during data collection at beamline 4A of Pohang Light Source, Korea. H.J. is supported by Grant M10420010001-04N2001-00110 from MOST, Korea and by the Molecular Imaging Program at the Korea Institute of Science and Technology. K.Y.H. is supported by the Functional Proteomics Center, 21C Frontier Program of the Korea Ministry of Science and Technology. Copyright: Copyright 2009 Elsevier B.V., All rights reserved.. PY - 2007/3/23. Y1 - 2007/3/23. N2 - The photoswitching behavior of green fluorescent proteins (GFPs) or GFP-like proteins is increasingly recognized as a new technique for optical marking. Recently, ...
The green fluorescent protein (GFP) is the most commonly used reporter protein for monitoring gene expression and protein localization in a variety of living and fixed cells, including not only prokaryotes, but eukaryotes also, e. EGFP inhibited both cell nest and growth development, and activated cell loss of life in Ku80-lacking hamster cells, i.y., xrs-6 cells. In addition, Ku80 attenuated EGFP-induced cytotoxicity in the xrs-6 cells. No EGFP-induced cytotoxicity was noticed in the NHEJ primary proteins XRCC4-lacking hamster cells, i.y., XR-1 cells. Furthermore, Substantially enhanced X-ray-induced cytotoxicity in the xrs-6 cells EGFP. These outcomes recommend that Ku80 has a essential function in the story NHEJ-independent protection system against EGFP-induced cytotoxicity. Extreme care should end up being used in taking into consideration of the potential impact by the tension response system, specifically, the Ku80-reliant reduction system of EGFP-induced cytotoxicity, getting turned on, ...
Mechanics of living mammalian cytoplasm 1.Overview The cytoplasm of living mammalian cells is a crowded, yet dynamic environment(1). It provides the key physical environment to the cellular factory and all intracellular physiological processes from transcription, translation, to protein binding and folding. Therefore, understanding the fundamental physical nature of the cytoplasm is critical to understanding the basic physiology of cells. Moreover, there are continuous intracellular movements that are vital for cell function, such as transport of vesicles and other organelles. While biological motors and other enzymatic processes provide key driving forces for these activities, the mechanical behavior of the cytoplasm are crucial for determining the mechanical resistance that cellular compartments experience. Both the active driving force and appropriate mechanical environment are critical for shaping the living cellular machinery. However, while the force that molecular motors generate both ...
The culture of human osteosarcoma cells featured in this section was transfected with a pDsRed-Mitochondria plasmid subcellular localization vector, thus localizing a red fluorescent protein tag to the intracellular mitochondrial network.
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Photoswitchable protein IrisFP molecule. Computer model showing the structure of the reversibly photoswitchable green to red fluorescent protein IrisFP. - Stock Image C035/6331
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CH Citrines EZr SedDanDun. A vertical pedigree lists the siblings of each dog in the pedigree. Siblings are found by searching for dogs in the database with the same parents. They may be from the same litter or from another breeding of the same parents. In most cases, this pedigree will be incomplete, as not all dogs have all their siblings entered in the database.. Vertical pedigree: ...
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We report for the first time the rela time non-invasive kinetic analysis of three steps in the NF-κB signalling pathway; IκBα degradation, p65 translocation and NF-κB-dependent transcription. We have used these tools to investigate the link between the kinetics of the NF-κB pathway, the levels of NF-κB and IκB proteins in cell compartments, and the resulting timing of transcription.. We showed that both the p65-EGFP and p65-dsRed fluorescent fusion proteins gave rise to the nuclear translocation in response to TNFα treatment, which is characteristic of the functional endogenous protein. Ding et al. previously reported an endogenous p65 nuclear translocation half time of 7-8 minutes in HeLa cells following TNFα stimulation ( Ding et al., 1998). In comparison, we obtained a longer half time of 19±2.9 minutes for nuclear translocation of p65-EGFP in singly transfected cells ( Fig. 1A) in agreement with other studies using a p65-EGFP fluorescent fusion protein and stimulation with IL-1β ( ...
The collection of specimens illustrated in this section demonstrates the effectiveness of the Nikon YFP HYQ filter combination with a series of cell lines containing a chimeric EYFP plasmid vector that expresses a fluorescent fusion protein targeted at the intracellular Golgi apparatus.
The collection of specimens illustrated in this section demonstrates the effectiveness of the Nikon YFP HYQ filter combination with a series of cell lines containing a chimeric EYFP plasmid vector that expresses a fluorescent fusion protein targeted at the cellular endosomal network.
pKatushka2S-N is a mammalian expression vector encoding far-red fluorescent protein Katushka2S (see reporter description). The vector allows generation of fusions to the Katushka2S N-terminus and expression of Katushka2S fusions or Katushka2S alone in eukaryotic (mammalian) cells. Katushka2S codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the Katushka2S coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between PCMV IE and Katushka2S coding sequence. The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3-end of the ...
Spectral imaging and linear unmixing has become an important tool in confocal and widefield fluorescence microscopy to discriminate between fluorophores having overlapping spectral characteristics.
Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol...
Congratulations to Dr Mark Fricker for his work on the paper ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology that was published open access on the eLIFE website.. Read the paper here: https://doi.org/10.7554/eLife.26770. Press release: https://www.uni-bonn.de/news/178-2017. ...
In search of localised membrane protein assembly centres in bacteria. PspA (Phage-Shock Protein A) is a widespread bacterial protein known to be important for preserving membrane integrity under environmental stress conditions. The related Vipp1 protein is found in cyanobacteria (and plant chloroplasts) and seems to play a crucial role in the biogenesis of photosynthetic membranes. In both case, the mechanism of action of the protein is enigmatic. We have visualised both proteins in vivo using fluorescence microscopy and fluorescent protein tags, and found that they form clusters near to the membrane under the conditions when they are likely to be physiologically active. Combined with biochemical identification of interaction partners under these conditions, this suggests a novel hypothesis: that both these proteins may help to organise assembly centres for the rapid and localised production of membrane and secreted proteins. A key prediction of this hypothesis is that specific mRNA molecules ...