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

Aaron Kolb // Brandt Lab // Publications // Dec 06 2003. PubMed ID: 14656463. Author(s): Kolb AW, Brandt CR. Enhanced isolation of low frequency herpes simplex virus recombinants using green-fluorescent protein and FACS. J Virol Methods. 2004 Jan;115(1):73-81. Journal: Journal Of Virological Methods, Volume 115, Issue 1, Jan 2004. The generation of recombinant herpes simplex virus to study the effect of engineered mutations on viral biology relies on the isolation of recombinants from a mixed population of viruses following a marker transfer procedure. Currently, the E. coli lacZ or green-fluorescent protein (GFP) genes are most frequently used as markers for isolation and isolation of recombinants relies on visual screening of plaques. Alternatively, novel restriction site changes can be inserted into a gene followed by screening of individual plaques for the novel change. These methods are inefficient when the frequency of recombinants in the pool of viruses is low. Using GFP as a selection ...

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 ...

Molecular Plant-Microbe Interactions 10:394-400...Continual Green-Fluorescent Protein Monitoring of Cauliflower Mosaic Virus 35S Promoter Activity in Nematode-Induced Feeding Cells in Arabidopsis thaliana...

Read this Science Presentation or Speech and over 30,000 other research documents. Introduction to Green Fluorescent Protein (gfp).. Introduction to Green Fluorescent Protein (GFP) The green fluorescent protein (GFP) obtained from the jellyfish Aequorea victoria has become one of the most widely researched proteins in biochemistry and cell biology, In early 1960s researcher named Osamu Shimomura set out to find the reason for this luminescence , after harvesting...

OV-IA82Δ113 and OV-IA82Δ116 viruses were constructed by infecting OFTu cells (in T25 flasks) at a multiplicity of infection (MOI) of 1.0 with wild type OV-IA 82 for 3 hours and subsequently transfecting the cells with 10 μg of pSVP-113LF-EGFP-113RF and pSVP-116LF-EGFP-116RF transfer vectors by standard in vivo recombination protocols [28, 29]. Transfections were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers instructions.. Viruses were harvested 48 h pi by scraping infected/transfected OFTu cells into sterile 15 ml conical tubes. The cell suspensions were vortexed, frozen/thawed 3 times, and then centrifuged at 1000 rpm, for 10 min at 4°C (Eppendorf Centrifuge 5810R, 15 amp version, Hamburg, Germany). The supernatant (viruses) were transferred into 2 ml cryogenic vials (Corning, NY, USA) and stored at -80°C for future use.. In order to select and purify recombinant viruses, limited dilution and plaque assays were performed. 3 ×104 of ...

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 ...

The monoclonal Anti-GFP-HRP antibody facilitates the fast and convenient detection of GFP and GFP derivatives or GFP-tagged fusion proteins in Western blot or ELISA applications. Conjugation to horseradish peroxidase (HRP) simplifies Western blot or ELISA analysis as incubation with secondary antibodies becomes dispensable. After incubation the Anti-GFP-HRP antibody can be directly detected using commercially available chemiluminescent reagents. The monoclonal Anti-GFP antibody specifically reconizes wild-type GFP from Aequorea victoria and derivatives, e.g., EGFP, CFP, YFP, and BFP. - 中国

Expression and Purification of recombinant Green Fluorescent Protein ABSTRACT: The purpose of this experiment was to determine if a His-6 tagged recombinant form of Green Fluorescent Protein could be expressed in a pRSETA vector of E. Coli. This was determined through multiple procedures beginning with purifying the sample with Ni +2 agarose chromatography which showcased the relative fluorescent activity of the samples, which elution sample two (E2) had approximately 100,592.2 RFU/mg . The yield of total protein was found by use of a Bradford Assay and a standard curve. The purity of the GFP was determined by comparing the intensity of bands that appeared at around 31.4 kDa (the molecular weight of rGFP) to a molecular weight ladder on an SDS-PAGE gel. The Western Blot test, utilizing a nitrocellulous membrane, confirmed the expression of rGFP. The Western Blot confirmed that the correct bands were analyzed in the SDS-PAGE gel which E3 had an estimated purity of 0.4, indicating a yield of ...

Goat Polyclonal GFP antibody for FLISA, ICC, FACS, IHC (fro), IF, PLA, WB. Published in 7 Pubmed References. Order this anti-GFP antibody. | Product number ABIN151299

TY - JOUR. T1 - A transgenic rat expressing green fluorescent protein (GFP) in peripheral nerves provides a new hindlimb model for the study of nerve injury and regeneration. AU - Moore, Amy M.. AU - Borschel, Gregory H.. AU - Santosa, Katherine B.. AU - Flagg, Eric R.. AU - Tong, Alice Y.. AU - Kasukurthi, Rahul. AU - Newton, Piyaraj. AU - Yan, Ying. AU - Hunter, Daniel A.. AU - Johnson, Philip J.. AU - Mackinnon, Susan E.. PY - 2012/2/1. Y1 - 2012/2/1. N2 - Background: In order to evaluate nerve regeneration in clinically relevant hindlimb surgical paradigms not feasible in fluorescent mice models, we developed a rat that expresses green fluorescent protein (GFP) in neural tissue. Methods: Transgenic Sprague-Dawley rat lines were created using pronuclear injection of a transgene expressing GFP under the control of the thy1 gene. Nerves were imaged under fluorescence microscopy and muscles were imaged with confocal microscopy to determine GFP expression following sciatic nerve crush, ...

Anti-GFP antibody conjugated to Biotin [LGB-1] validated for WB. Referenced in 1 publication. Immunogen corresponding to recombinant full length protein

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.

Lack of expression of dystrophin leads to degeneration of muscle fibers and infiltration of connective and adipose tissue. Cell transplantation therapy has been proposed as a treatment for intractable muscle degenerative disorders. Several reports have demonstrated the ability of bone-marrow derived cells (BMDC) to contribute to non-haematopoietic tissues including epithelium, heart, liver, skeletal muscle and brain following transplantation by means of fusion and reprogramming. A key issue is the extent to which fusion and reprogramming can occur in vivo, particularly under conditions of myogenic deterioration.To investigate the therapeutic potential of bone marrow transplantation in monogenetic myopathy, green fluorescent protein-positive (GFP+) bone marrow cells were transplanted into non-irradiated c-kit receptor-deficient (W⁴¹) mdx mice. This model allows BMDC reconstitution in the absence of irradiation induced myeloablation. We provide the first report of BMDC fusion in a ...

Aims Cells derived from the stroma vascular fraction (SVF) of mouse adipose tissue can spontaneously give rise to rare, functional, cardiac-like cells in vitro. This study aimed to improve the production of adipose-derived cardiomyogenic cells (AD-CMG), to characterize them and to assess their cardiac fate and functional outcomes after their administration in a mouse model of acute myocardial infarction. Methods and results The culture process optimized to improve in vitro cardiac specification consisted of a primary culture of murine SVF cells in semi-solid methylcellulose medium, a selection of AD-CMG cell clusters, and a secondary culture and expansion in BHK21 medium. AD-CMG cells were CD29+, CD31−, CD34−, CD44+, CD45−, CD81+, CD90−, CD117−, and Flk-1− and expressed several cardiac contractile proteins. After 1, 2, and 4 weeks of their injection in mice having acute myocardial infarction, a strong presence of green fluorescent protein-positive cells was identified by ...

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP

Novel fluorescent tools such as green fluorescent protein analogs and Fluorogen Activating Proteins (FAPs) are useful in biological imaging to track protein dynamics in real-time with low fluorescence background. evolution experiments of both VH-VL M8 and M8VL, led us to rationally design tandem, covalent homodimers of M8VL domains joined by a flexible linker that have a higher affinity for DIR and great quantum yield. The introduction of fluorescent systems has revolutionized mobile imaging and molecular biology, as well as the electricity of encoded fluorescent proteins, such as for example green fluorescent proteins (GFP), for the recognition of particular proteins appealing can be well recorded (1). Theres a dependence on extra still, well-characterized tools offering real-time, high signal-to-noise fluorescence and demonstrate high fluorescence quantum produce (?f), photo-stability, and a wide spectral range. Fluorogen Activating Protein (FAPs) are section of book, immunoglobulin-based, ...

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 ...

TY - JOUR. T1 - Studying cytoskeletal dynamics in living cells using green fluorescent protein. AU - Yoon, Yisang. AU - Pitts, Kelly. AU - McNiven, Mark. PY - 2002/7/4. Y1 - 2002/7/4. N2 - Microfilaments, intermediate filaments, and microtubules are three major cytoskeletal systems providing cells with stability to maintain proper shape. Although the word cytoskeleton implicates rigidity, it is quite dynamic exhibiting constant changes within cells. In addition to providing cell stability, it participates in a variety of essential and dynamic cellular processes including cell migration, cell division, intracellular transport, vesicular trafficking, and organelle morphogenesis. During the past eight years since the green fluorescent protein (GFP) was first used as a marker for the exogenous gene expression, it has been an especially booming era for live cell observations of intracellular movement of many proteins. Because of the dynamic behavior of the cytoskeleton in the cell, GFP has ...

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). ...

PubMed comprises more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

The assessment of how many cells have been successfully transfected or transduced in a cell population is a basic and critical evaluation parameter in many cell and molecular biology labs. Commonly, the cells of interest are transfected or transduced with a construct that results in the expression of a fluorescent protein (FP) reporter, such as GFP.

TY - JOUR. T1 - Chapter 1. T2 - Biophysics of the Green Fluorescent Protein. AU - Prendergast, F. G.. PY - 1998/1/1. Y1 - 1998/1/1. N2 - It is almost certainly a truism that the interpretation of the fluorescence of a protein matrix-embedded chromophore in terms of the physicochemical character of its environment requires that the tertiary structure of the protein be known to high resolution. This reality derives from the complexity of the photophysics of most fluorescent molecules-complexity that reveals the imperfections of available theory. The accuracy of these dicta is highlighted by the biophysical properties of the green fluorescent protein (GFP) now being so elegantly elucidated from the application of X-ray crystallography, ultrafast optical spectroscopy, and site-specific mutagenesis. Given the apparent malleability of the GFP sequence and the sensitivity of the chromophores photophysics to a broad spectrum of physicochemical factors, it is inevitable that additional useful and ...

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, ...

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 ...

Clontech offers two improved green fluorescent proteins: AcGFP1 is a monomeric green fluorescent protein and ZsGreen1 is extremely bright.

Clontech offers two improved green fluorescent proteins: AcGFP1 is a monomeric green fluorescent protein and ZsGreen1 is extremely bright.

The gene encoding green fluorescent protein (GFP) has recently become an important visual marker of gene expression in eukaryotic organisms, as it is more sensitive than other reporter genes, requires no special cofactors for detection (7), and can be quantitated with a spectrofluorimeter (24). GFP has not been as widely applied to prokaryotic organisms because of a lack of constructs useful for diverse groups of bacteria, although GFP vectors are available for specialized bacterial systems (13, 24, 33, 41, 42). The wild-type gfpgene has been mutated to improve detection and expression of the fluorescent protein in prokaryotes (10, 18, 30), and both the wild-type and mutated forms have been used to construct less specialized bacterial GFP vectors.. A broad-host-range plasmid expressing the improved gfp(mut2) (10) gene from either a lac or annpt-2 promoter has been used successfully to tag gram-negative soil bacteria with GFP (27). Escherichia coli-Pseudomonas spp. shuttle vectors ...

TY - JOUR. T1 - Dynamics of insulin-stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein. AU - Dobson, SP. AU - Livingston, C. AU - Gould, GW. AU - Tavaré, JM. PY - 1996. Y1 - 1996. M3 - Article (Academic Journal). VL - 393. SP - 179. EP - 184. JO - FEBS Letters. JF - FEBS Letters. SN - 0014-5793. ER - ...

Sino Biologcial offers high quality green fluorescent protein GFPSpark® with features of bright green fluorescence, high pH-stability and fast maturation.

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.. ...

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.

UBC-GFP transgenic mice express green fluorescent protein directed by the human ubiqutin C promoter, and were discovered to have transgene insertion on chromosome 17 resulting in linkage to H-2|sup|b|/sup| MHC haplotype (see details below). Certain hematopoietic cell types display distinct expression levels of GFP, allowing identification of different cells types by FACS analysis. This strain is a useful tool for studying hematopoietic cell differentiation and in vivo leukocyte tracking. This transgene is also available on C57BL/6J as Stock No. |a href=https://www.jax.org/strain/004353|004353|/a|. |br /||br /||strong|In 2020, the UBC-GFP transgene was determined to have integrated at Chr17:29,435,589 - a noncoding region centromeric from the H-2 locus (~4.6 Mbp away from |em|H2-K1|/em|) - and is closely linked to H-2|sup|b|/sup| MHC haplotype. See Important Note for additional details|/strong|.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol...

Use of a dicistronic expression cassette encoding the green fluorescent protein for the screening and selection of cells expressing inducible gene products

A calculator mutagenesis technique has been used to characterize the structural effects associated with more than 46,000 amino acid variants simple and multiple green fluorescent proteins Aequorea ...

Green lights in the dark When someone first shows up in our lab, the prime goal I set up for him or her is to make green cells - I mean to introduce a Green Fluorescent Protein into a mammalian cell culture. In order to be able to perform this one has to know some basic molecular…

Shop a large selection of products and learn more about Thermo Scientific Lab Vision GFP (Green Fluorescent Protein) Ab-1, Mouse 100µL; 200µg/mL; Unlabeled; Purified

Photodamage of symbiotic algae exposed to thermal stress is involved in mass coral bleaching, a major cause of reef decline. Photoprotection is therefore a vital part of coral stress physiology. Corals produce a variety of green fluorescent protein (GFP)-like proteins from which some representatives screen the symbiotic algae from excess light. Different tissue concentrations of these GFP-like proteins distinguish colour morphs that are characteristic for many coral species. The question arises whether these pigmentation differences may diversify the niches that can be occupied by corals along the steep light gradient that structures coral reef communities. We assessed the implications of GFP-like protein expression in two color morphs of the symbiotic coral Hydnophora grandis, both associated with the same Symbiodinium sp. (subclade C40). The colour morphs of this species (high fluorescent, HF; and low fluorescent, LF), characterized by markedly different contents of a cyan fluorescent protein, were

In most previous papers concerning nisin quantification the authors emphasized the lowest detectable amount of nisin, which was given as the final assay concentration. However, this value seldom describes the most important numerical value giving true limits to the usefulness of the method in question, namely, the lowest detectable concentration of nisin in a food sample. Indeed, in some studies food material was spiked with amounts of nisin far from the linear dose-response range of the assay described, and the authors did not reveal the solvent into which the food extract was diluted prior to measurement (3, 5). Because of the sensitivity of immunological methods to interfering substances in a sample matrix, this kind of reporting makes it impossible for a reader to decide whether the assay in question is useful for his or her application.. At present, the most widely used quantification assay for nisin, the agar diffusion method, which was developed by Tramer and Fowler in 1964, is more ...

Green fluorescent protein (GFP), a 27 kDa protein derived from the jellyfish Aequorea victoria, emits green light (emission peak 509 nm) when excited by blue light (excitation peak 395 nm). GFP has become an invaluable tool in cell biology research, since its intrinsic fluorescence can be visualized in living cells. GFP fluorescence is stable under fixation conditions and suitable for a variety of applications. GFP has been widely used as a reporter for gene expression, enabling researchers to visualize and localize GFP-tagged proteins within living cells without the need for chemical staining. Other applications of GFP include assessment of protein-protein interactions through the yeast two hybrid system and measurement of distance between proteins through fluorescence energy transfer (FRET) protocols. GFP is used to measure single cell metastasis and successful proliferation of stem cells.. ...

A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms. ...

Fig. 4 Functional assays show increased transformation potential and sensitivity to TNK2 inhibition.. (A) Total colony formation in mouse bone marrow colony formation assay. Mouse bone marrow cells were cotransduced to express PTPN11, PTPN11 E76K, TNK2, or empty vector controls. Cells were selected for GFP+ (green fluorescent protein-positive) and puromycin resistance and plated in a methylcellulose GM-CSF sensitivity colony formation assay. Colonies were counted at 14 days [GM-CSF] = 0.05 nM (0.71 ng/ml). ****P , 0.0001 by one-way ANOVA. (B) Total colony formation in mouse bone marrow colony formation assay in cells transduced with PTPN11, PTPN11 E76K, or PTPN11 G60R. Cells were sorted for GFP+. Cells were plated with increasing concentrations of dasatinib. ***P , 0.005 and ****P , 0.0005 by one-way ANOVA. (C) Total colony formation and percent total colony formation in mouse bone marrow colony formation assay. Mouse bone marrow cells were cotransduced to express PTPN11 E76K and TNK2 or TNK2 ...

Magnetic resonance imaging (MRI) of magnetically labeled stem cells has become a valuable tool in the understanding and evaluation of experimental stem cell-based therapies of degenerative central nervous system disorders. This comprehensive study assesses the impact of magnetic labeling of both human and rodent stem cell-containing populations on multiple biologic parameters as maintenance of stemness and oxidative stress levels. Cells were efficiently magnetically labeled with very small superparamagnetic iron oxide particles. Only under the condition of tailored labeling strategies can the impact of magnetic labeling on vitality, proliferation, pluripotency, and oxidative stress levels be minimized. In a rat model of Parkinson disease, magnetically labeled mouse embryonic stem cells were tracked by high-field MRI for 6 months. Significant interindividual differences concerning the spatial distribution of cells became evident. Histologically, transplanted green fluorescent protein-positive ...

Neuropeptide S (NPS) has been associated with a number of complex brain functions, including anxiety-like behaviors, arousal, sleep-wakefulness regulation, drug-seeking behaviors, and learning and memory. In order to better understand how NPS influences these functions in a neuronal network context, it is critical to identify transmitter systems that control NPS release and transmitters that are co-released with NPS. For this purpose, we generated several lines of transgenic mice that express enhanced green-fluorescent protein (EGFP) under control of the endogenous NPS precursor promoter. NPS/EGFP-transgenic mice show anatomically correct and overlapping expression of both NPS and EGFP. A total number of similar to 500 NPS/EGFP-positive neurons are present in the mouse brain, located in the pericoerulear region and the Kolliker-Fuse nucleus. NPS and transgene expression is first detectable around E14, indicating a potential role for NPS in brain development. EGFP-positive cells were harvested by ...

Funded by the NIH National Center for Advancing Translational Sciences through its Clinical and Translational Science Awards Program, grant number UL1TR002541 ...

Spinal cord injuries (SCI) are disastrous neuropathologies causing permanent disabilities. The availability of different strains of mice is valuable for studying the pathophysiological mechanisms involved in SCI. However, strain differences have a profound effect on spontaneous functional recovery after SCI. CX3CR1+/eGFP and Aldh1l1-EGFP mice that express green fluorescent protein in microglia/monocytes and astrocytes, respectively, are particularly useful to study glial reactivity. Whereas CX3CR1+/eGFP mice have C57BL/6 background, Aldh1l1-EGFP are in Swiss Webster background. We first assessed spontaneous functional recovery in CX3CR1+/eGFP and Aldh1l1-EGFP mice over 6 weeks after lateral spinal cord hemisection. Second, we carried out a longitudinal follow-up of lesion evolution using in vivo T2-weighted magnetic resonance imaging (MRI). Finally, we performed in-depth analysis of the spinal cord tissue using ex vivo T2-weighted MRI as well as detailed histology. We demonstrate that CX3CR1+/eGFP mice

TY - JOUR. T1 - Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein. AU - Sun, Duxin. AU - Landowski, Christopher P.. AU - Chu, Xiaoyan. AU - Wallsten, Richard. AU - Komorowski, Thomas E.. AU - Fleisher, David. AU - Amidon, Gordon L.. PY - 2001/12/1. Y1 - 2001/12/1. N2 - An hPepT1-GFP fusion construct was made to study drug inhibition of dipeptide uptake and apical, basolateral, or subcellular hPepT1 localization. The hPepT1 stop codon was mutated by polymerase chain reaction and was subsequently cloned into the pEGFP-N1 vector. The hPepT1-GFP fusion construct was then transfected into Caco-2 and HeLa cells, and drug inhibition was studied by inhibiting 3H-Gly-Sar uptake. Western blot analysis was used to determine hPepT1-GFP expression levels and confocal microscopy was used to examine the localization. Both anti-hPepT1 antibody and anti-GFP antibody recognized a 120kd hPepT1-GFP fusion protein in the transfected cells. The 3H-Gly-Sar uptake in transfected ...

Green fluorescent protein (GFP), molecular model. The molecule has a cylindrical structure formed from beta sheets (ribbons). GFP is found in the Pacific jellyfish Aequorea victoria. It fluoresces green when blue light is shone on it. GFP is widely used as a research tool in biology and medicine. The gene coding for it can be tagged to the genes of other proteins or viruses to study their movements within cells. They can also be used to tag cancer cells to track their spread through the body. - Stock Image F006/9343

Purpose : The purpose of the current study is to determine in-depth functions of selected transcripts that are enriched in rod photoreceptors, in order to gain insights into intrinsic mechanisms underlying rod development and regeneration. Methods : We used a transgenic zebrafish line (XOPS:eGFP) in which rod photoreceptors express green fluorescent protein (GFP) as a model organism for this study. RNA-seq of FACS-sorted dissociated retinal cells was performed to identify differentially expressed (in GFP+ vs. GFP- cells) transcripts with FDR , 0.01. Selected rod-specific genes were prioritized for further qRT-PCR and in situ hybridization studies at different life stages of the zebrafish, and for qRT-PCR studies of rods that regenerated after widespread chemical lesioning of the retina. The rxrγa gene was of particular interest as its transcript was enriched in rods, while previous studies in mouse indicated roles in cone determination. Therefore, a new rxrγa mutant line was created by ...

TY - JOUR. T1 - Probing intra-molecular mechanics of single circularly permuted green fluorescent protein with atomic force microscopy. AU - Wang, Tong. AU - Nakajima, Ken. AU - Miyawaki, Atsushi. AU - Hara, Masahiko. PY - 2005/11/1. Y1 - 2005/11/1. N2 - We investigated the mechanical unfolding of single circularly permuted green fluorescent protein (cpGFP) with atomic force microscopy (AFM). The molecule was stretched from its N- and C-termini by an external force causing an elongation of the polypeptide chain up to its full length. The features of the force-extension (F-E) curves were found to depend on the stretching speed. At fast speeds, we detected one peak in the F-E curves before final rupture of the extended molecule, which we interpreted as the unfolding of two terminal halves within cpGFP. We observed several more force peaks in a sawtooth pattern at much slower speeds, and explained the appearance of such force peaks as cooperative unfolding of the hidden sub-structures inside each ...

Hi, brief question. does anti-GFP from invitrogen can recognize EGFP from clontech? I am currently trying to detect EGFP on western blot by using anti-GFP from invitrogen but I cannot detect it. Also anybody working with anti-GFP from invitrogen and having any comment? should I use anti-GFP from other company? mUsClemUsClemUsClemUsClemUsCle Keitaro YAMANOUCHI D. V. M., Ph. D. Muscle Biology Group Nutritional Science University of Arizona 601 Shantz Building Tucson AZ 85721-0038 Tel: 520-621-3829 Fax: 520-621-1396 e-mail: keitaro at ag.arizona.edu mUsClemUsClemUsClemUsClemUsCle ...

In the present paper, we introduce a transgenic mouse line whose sperm express green fluorescent protein (GFP) in their acrosome and red fluorescent protein (RFP) in their mitochondria [B6D2F1- Tg(CAG/su9-DsRed2, Acr3-EGFP)RBGS002Osb]. The dual fluorescent sperm showed normal fertilizing ability in …

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 ...

Light micrograph (LM Fluorescence) of a transformed bacteria colony (Escherichia coli) containing a jellyfish gene for GFP (green fluorescent protein). GFP is a 238 amino acid protein found in the Pacific jellyfish (Aequorea victoria). It exhibits a bright green fluorescence (green bioluminescence) when exposed to ultraviolet blue light. GFP is widely used as a research tool in biology and medicine. The gene coding for GFP can be tagged to the genes of other proteins or viruses to study their movements within cells. GFP can also be used to tagged cancer cells to track their spread through the body. The purpose of both bioluminescence and GFP fluorescence in jellyfish is unknown. Magnification: x20 when shortest axis printed at 25 millimetres. - Stock Image C032/2759

Over the past several years, we have differentiated green fluorescent protein-expressing human embryonic stem cells (GFP-hESC) into mesenchymal stem cell-like cells (eMSCs). These eMSCs expressed markers (CD-29, CD-44, CD-73, CD-105, CD-166 and Nestin, but not CD-34, C45, CD-106, SSEA-4 or Oct-4) which are consistent with a mesenchymal stem cell state. We have transplanted eMSCs into the femoral veins of SHRs following MCAO. The expression of GFP allows us to follow the migration and further differentiation of the eMSCs in the ischemic brain. We observed migration of the injected eMSCs to the infarction region (Fig. 1) and their subsequent differentiation into neurons (which expressed β-tubulin III, MAP2, HuC and neurofilament) (see Fig. 2) and vascular endothelial cells (which expressed vWF; see Fig. 3). The grafted cells do not appear to differentiate into astrocytes.. ...

Over the past several years, we have differentiated green fluorescent protein-expressing human embryonic stem cells (GFP-hESC) into mesenchymal stem cell-like cells (eMSCs). These eMSCs expressed markers (CD-29, CD-44, CD-73, CD-105, CD-166 and Nestin, but not CD-34, C45, CD-106, SSEA-4 or Oct-4) which are consistent with a mesenchymal stem cell state. We have transplanted eMSCs into the femoral veins of SHRs following MCAO. The expression of GFP allows us to follow the migration and further differentiation of the eMSCs in the ischemic brain. We observed migration of the injected eMSCs to the infarction region (Fig. 1) and their subsequent differentiation into neurons (which expressed β-tubulin III, MAP2, HuC and neurofilament) (see Fig. 2) and vascular endothelial cells (which expressed vWF; see Fig. 3). The grafted cells do not appear to differentiate into astrocytes.. ...

Abstract: Background: Sterile α motif and HD domain-containing protein-1 (SAMHD1) inhibits HIV-1 reverse transcription by decreasing the pool of intracellular deoxynucleotides. SAMHD1 is controlled by cyclin-dependent kinase (CDK)-mediated phosphorylation. However, the exact mechanism of SAMHD1 regulation in primary cells is unclear. We explore the effect of palbociclib, a CDK6 inhibitor, in HIV-1 replication.. Methods: Human primary monocytes were differentiated into macrophages with monocyte-colony stimulating factor and CD4+ T lymphocytes stimulated with phytohaemagglutinin (PHA)/interleukin-2. Cells were treated with palbociclib and then infected with a Green fluorescent protein-expressing HIV-1 or R5 HIV-1 BaL. Viral DNA was measured by quantitative PCR and infection assessed by flow cytometry. Deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method.. Results: Pan-CDK inhibitors AT7519, roscovitine and purvalanol A reduced SAMHD1 phosphorylation. HIV-1 ...

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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 ...

Need antibody products for research? Find and compare multiple sources of anti-Green Fluorescent Protein (GFP) antibody using the Linscotts Directory search engine. Monoclonal, polyclonal, and recombinant antibodies. Select applications, conjugates, hosts, and reactivity. Get complete supplier details here.

Need antibody products for research? Find and compare multiple sources of anti-Green Fluorescent Protein (GFP) antibody using the Linscotts Directory search engine. Monoclonal, polyclonal, and recombinant antibodies. Select applications, conjugates, hosts, and reactivity. Get complete supplier details here.

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.

Transgenic techniques have rapidly evolved in recent years. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. The purpose of this study was to assess in vitro development, transgene expression, and integration following pronuclear or cytoplasmic microinjection of condensed or linear green fluorescent protein DNA into murine embryos using electroporation. In experiment 1, the effect of embryo orientation (group or linear) within the electroporation chamber on development was evaluated using zygotes which received one pulse duration (10 msec), and one of two voltages (250 or 400 V). Zygotes that received 400 V had the lowest development score (Group, 2.06 ? 0.12; Linear, 1.97 ? 0.13), irrespective of orientation. Embryos that received 250 V had the highest development of the voltage treated groups (Group 3.42 ? 0.12; Linear 3.32 ? 0.12), irrespective of orientation, and development was lower than the control embryos (Control 4.28 ? 0.12; Mannitol ...

Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein (GFP) fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) at yields of ≥10 mg/L, following

Bone marrow harbors cells that have the capacity to differentiate into cells of nonhematopoietic tissues of neuronal, endothelial, epithelial, and muscular phenotype. Here we demonstrate that bone marrow-derived cells populate pancreatic islets of Langerhans. Bone marrow cells from male mice that express, using a CRE-LoxP system, an enhanced green fluorescent protein (EGFP) if the insulin gene is actively transcribed were transplanted into lethally irradiated recipient female mice. Four to six weeks after transplantation, recipient mice revealed Y chromosome and EGFP double-positive cells in their pancreatic islets. Neither bone marrow cells nor circulating peripheral blood nucleated cells of donor or recipient mice had any detectable EGFP. EGFP-positive cells purified from islets express insulin, glucose transporter 2 (GLUT2), and transcription factors typically found in pancreatic β cells. Furthermore, in vitro these bone marrow-derived cells exhibit - as do pancreatic β cells - ...

P. Didier, L. Guidoni, and E. Weiss, Ultrafast Excited-state Dynamics of Genetic Fusions: The Green Fluorescent Protein as a Folding Reporter, in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science Conference and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, 2006), paper QMH1 ...

A reporter gene assay using the human placental secreted alkaline phosphatase (SEAP) or the green fluorescent protein for G proteiencoupled receptors

Neurons and glia are derived from a common set of precursor stem cells. Morrow et al. used a cortical slice assay to examine the signals required to specify neuronal versus glial cell fate and differentiation. Dissociated embryonic neural stem cells from a green fluorescent protein (GFP)-expressing strain of mice were plated with cortical slices from mice of different ages (embryonic through postnatal). Cell fate and differentiation were monitored by changes in the morphology and expression of marker proteins for neurons and glia. Incubation of neural stem cells with embryonic cortical slices led to the development of mostly neuronal cells and incubation with postnatal cortical slices produced mostly glial cells, suggesting that different factors are produced at different stages of development and that the stem cells are poised to respond to either set of signals. Analysis of clones of the GFP-expressing cells showed that the signals were predominantly instructive and not trophic signals. The ...

We combine Fluorescence Recovery After Photobleaching (FRAP) experiments with mathematical modelling to study the dynamics inside the nucleus of both the TGF-β-sensitive transcriptional regulator Smad2, and Green-Fluorescent Protein (GFP). We show how combining modelling with bleaching strips of different areas allows a rigorous test of whether or not a protein is moving via diffusion as a single species. As noted recently by others, it is important to consider diffusion during the bleaching process. Neglecting it can cause serious error. Also, it is possible to use the bleaching process itself to provide an extra consistency test to the models predicting the recovery. With our method we show that the dynamics of GFP are consistent with it diffusing as a single species in a uniform environment in which flow is negligible. In contrast, the dynamics of the intracellular signal transducer Smad2 are never consistent with it moving as a single species via simple diffusion in a homogeneous ...

Mono- and Stereopictres of 5.0 Angstrom coordination sphere of Copper atom in PDB 1kyr: Crystal Structure of A Cu-Bound Green Fluorescent Protein Zn Biosensor

Fingerprint Dive into the research topics of Sensitivity of the yellow variant of green fluorescent protein to halides and nitrate [2]. Together they form a unique fingerprint. ...

The advent of jellyfish green fluorescent protein and its spectral variants, together with promising new fluorescent proteins from other classes of the Cnidarian phylum (coral and anemones), has greatly enhanced and promises to further boost the detection and localization of proteins in cell biology. It has been less widely appreciated that highly sensitive methods have also recently been developed for detecting the movement and localization in living cells of the very molecules that precede proteins in the gene expression pathway, i.e. RNAs. These approaches include the microinjection of fluorescent RNAs into living cells, the in vivo hybridization of fluorescent oligonucleotides to endogenous RNAs and the expression in cells of fluorescent RNA-binding proteins. This new field of fluorescent RNA cytochemistry is summarized in this article, with emphasis on the biological insights it has already provided. These new techniques are likely to soon collaborate with other emerging approaches to advance the

Sandia National Laboratories researchers are drawing inspiration from neurons in the brain, such as these green fluorescent protein-labeled neurons in a 1f52c read all about

Our researchers have developed a new technology that allows for the study of protein structure/folding/functions in the living cells. This new technology involves expressing a protein using bacteria and labeling the protein with probes. Researchers can then modify the recombinant protein with a special reagent that allows for the transfer of the modified protein into the living mammalian cells. This will also allow for the study of protein traffic in the cells. Furthermore, this new technology permits high-resolution structural biology techniques to be combined with cell biology techniques and provides a foundation for future applications of protein transduction technology, atomic resolution cell biology, and protein drug therapy to treat human disease. ...

Stem cells have great potential value for treating a number of diseases and conditions, including diabetes, Parkinsons, and spinal cord injuries. Applying stem cells for therapeutic purposes will require an in-depth understanding of their biology, not only of the genes they express, but also the functions of the proteins encoded by the genes. The goal of the project presented in this thesis was to develop a method for high-throughput analyses of protein localization in mouse stem cells. Localization information can provide insight into the functions and biological roles of proteins. ,br /,,br /, One means of studying protein localization involves creating proteins with a green fluorescent protein (GFP) reporter gene and analyzing their localization using fluorescence microscopy. The research outlined in this thesis focused on developing a system to create a large number of GFP-tagged proteins by constructing a cDNA?GFP fusion library. This involved exploring methods for optimizing cDNA ...

The effects of embryonic and larval ethanol exposure on brain development were visually monitored using transgenic zebrafish expressing cell-specific green fluorescent protein (GFP) marker genes. Specific subsets of GFP-expressing neurons were highly sensitive to ethanol exposure, but only during defined developmental windows. In the med12 mutant, which affects the Mediator co-activator complex component Med12, exposure to lower concentrations of ethanol was sufficient to reduce GFP expression in transgenic embryos. In transgenic embryos and larva containing GFP driven by an oxytocin-like (oxtl) promoter, ethanol exposure dramatically up-regulated GFP expression in a small group of hindbrain neurons, while having no effect on expression in the neuroendocrine preoptic area.. ...

A special protein found in jellyfish called green fluorescent protein (or GFP) glows green when a particular wavelength of light is shone on it. By following the green glow you know where GFP is. Can this help locate other proteins? GFP is expressed from a GFP gene and, like all genes, the GFP gene is made up of A, T, C and G subunits. This is important; it means that a gene from a person (e.g. the gene we found mutated in the Parkinsons family) can be attached to the jellyfish GFP gene to form a hybrid gene and therefore a hybrid protein: one half human and the other half jellyfish. Therefore, wherever the human protein goes the GFP protein goes too; when an expression plasmid containing the GFP hybrid gene is introduced into cells a particular part of the cell will glow green, demonstrating the human protein does its job there. ...

These tools will help advance any experiments utilizing Green Fluorescent Protein or Red Fluorescent Protein fusion constructs.. ...

Kicking off the evening was John Lee, from the University of Georgia, with his ambitiously titled talk Bioluminescence: The First 3000 Years. After a historical introduction to the long running observation of bioluminescence, via the discovery in 1672 that oxygen was necessary for bacterial luminescence, John told us how it was determined that bioluminescence is an enzyme mediated chemical reaction involving luciferase and luciferine. In the modern age of biochemistry it was determined that ATP is the substrate in this reaction. Following the elucidation of the structure of firefly luciferase in 1959, modern techniques (i.e. picosecond dynamic fluorescence spectroscopy and NMR) have allowed researchers to uncover the enzymes and processes involved in bioluminescence. One of the most important of these enzymes Green-fluorescent protein (GFP) was discovered in jellyfish by Shimomura (who evidently has a lab at his house!) and led to his Nobel prize in 2008. Due to GFPs widespread use in ...

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