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Patterns of Green Fluorescent Protein Expression in Transgenic Plants
Color profile: Generic CMYK printer profile Composite Default screen Plant Molecular Biology Reporter 18: 141a–141i, 2000 © 2000 International Society for Plant Molecular Biology. Printed in Canada. Publish by Abstract Patterns of Green Fluorescent Protein Expression in Transgenic Plants BRIAN K. HARPER1 and C. NEAL STEWART JR.2,* 1Novartis Agricultural Biotechnology Research, Inc., 3054 Cornwallis Rd., Research Triangle Park, NC 27709; 2Dept of Biology, University of North Carolina, Greensboro, NC 27402-6174 Abstract. Modified forms of genes encoding green fluorescent protein (GFP) can be mac- roscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP vi- sualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing trans- genic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice. Key words: expression patterns, green fluorescent protein, marker genes Patterns of IntroductionGFP expression Harper and Stewart Since the discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria it has become a frequently used tool in biology. -
Supplementary Materials and Method Immunostaining and Western Blot
Supplementary Materials and Method Immunostaining and Western Blot Analysis For immunofluorescence staining, mouse and human cells were fixed with 4% paraformaldehyde- PBS for 15 min. Following Triton-X100 permeabilization and blocking, cells were incubated with primary antibodies overnight at 4°C following with Alexa 594-conjugated secondary antibodies at 4°C for 1 hour (Thermo Fisher Scientific, 1:1000). Samples were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and immunofluorescence was detected using Olympus confocal microscopy. For western blot analysis, cells were lysed on ice using RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma). Primary Antibodies for Immunostaining and Western Blot Analysis: Yap (14074, Cell Signaling), pYAP (4911, Cell Signaling), Lats1 (3477, Cell Signaling), pLats1( 8654, Cell Signaling), Wnt5a (2530, Cell Signaling), cleaved Caspase-3 (9661, Cell Signaling), Ki-67 (VP-K451, Vector Laboratories), Cyr61 (sc-13100, Santa Cruz Biotechnology), CTGF (sc-14939, Santa Cruz Biotechnology), AXL (8661, Cell Signaling), pErk (4376, Cell Signaling), pMEK (4376, Cell Signaling), Ck-19 (16858-1-AP, Proteintech), Actin (A2228, Sigma Aldrich), Vinculin (V4139, Sigma Aldrich), Kras (sc-30, Santa Cruz Biotechnology). Ectopic expression of YAP1 and WNT5A in mouse and human cells To generate YAP1S127A-expressing stable Pa04C cells, Pa04C cells were transfected with a linearized pcDNA3.1 plasmid with or without YAP1 cDNA containing S127A substitution. Two days post-transfection using Lipofectamine1000, cultures were selected in G418 (Sigma) and single clones were picked and expanded for further analysis. Overexpression of YAPS127A or WNT5A in human or mouse cells other than Pa04C were acheieved with lentivral infection. Briefly, lentivirus infection was performed by transfecting 293T cells with either GFP control, YAP1S127A, or WNT5A cloned in pHAGE lentivirus vector {EF1α promoter-GW-IRES-eGFP (GW: Gateway modified)}. -
Analysis of Proteins by Immunoprecipitation
Laboratory Procedures, PJ Hansen Laboratory - University of Florida Analysis of Proteins by Immunoprecipitation P.J. Hansen1 1Dept. of Animal Sciences, University of Florida Introduction Immunoprecipitation is a procedure by which peptides or proteins that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics (molecular weight, isoelectric point, etc.). As usually practiced, the name of the procedure is a misnomer since removal of the antigen from solution does not depend upon the formation of an insoluble antibody-antigen complex. Rather, antibody-antigen complexes are removed from solution by addition of an insoluble form of an antibody binding protein such as Protein A, Protein G or second antibody (Figure 1). Thus, unlike other techniques based on immunoprecipitation, it is not necessary to determine the optimal antibody dilution that favors spontaneously-occurring immunoprecipitates. Figure 1. Schematic representation of the principle of immunoprecipitation. An antibody added to a mixture of radiolabeled (*) and unlabeled proteins binds specifically to its antigen (A) (left tube). Antibody- antigen complex is absorbed from solution through the addition of an immobilized antibody binding protein such as Protein A-Sepharose beads (middle panel). Upon centrifugation, the antibody-antigen complex is brought down in the pellet (right panel). Subsequent liberation of the antigen can be achieved by boiling the sample in the presence of SDS. Typically, the antigen is made radioactive before the immunoprecipitation procedure, either by culturing cells with radioactive precursor or by labeling the molecule after synthesis has been completed (e.g., by radioiodination to iodinate tyrosine residues or by sodium [3H]borohydride reduction to label carbohydrate). -
The Immunoassay Guide to Successful Mass Spectrometry
The Immunoassay Guide to Successful Mass Spectrometry Orr Sharpe Robinson Lab SUMS User Meeting October 29, 2013 What is it ? Hey! Look at that! Something is reacting in here! I just wish I knew what it is! anti-phospho-Tyrosine Maybe we should mass spec it! Coffey GP et.al. 2009 JCS 22(3137-44) True or false 1. A big western blot band means I have a LOT of protein 2. One band = 1 protein Big band on Western blot Bands are affected mainly by: Antibody affinity to the antigen Number of available epitopes Remember: After the Ag-Ab interaction, you are amplifying the signal by using an enzyme linked to a secondary antibody. How many proteins are in a band? Human genome: 20,000 genes=100,000 proteins There are about 5000 different proteins, not including PTMs, in a given cell at a single time point. Huge dynamic range 2D-PAGE: about 1000 spots are visible. 1D-PAGE: about 60 -100 bands are visible - So, how many proteins are in my band? Separation is the key! Can you IP your protein of interest? Can you find other way to help with the separation? -Organelle enrichment -PTMs enrichment -Size enrichment Have you optimized your running conditions? Choose the right gel and the right running conditions! Immunoprecipitation, in theory Step 1: Create a complex between a desired protein (Antigen) and an Antibody Step 2: Pull down the complex and remove the unbound proteins Step 3: Elute your antigen and analyze Immunoprecipitation, in real life Flow through Wash Elution M 170kDa 130kDa 100kDa 70kDa 55kDa 40kDa 35kDa 25kDa Lung tissue lysate, IP with patient sera , Coomassie stain Rabinovitch and Robinson labs, unpublished data Optimizing immunoprecipitation You need: A good antibody that can IP The right beads: i. -
Wo2015188839a2
Downloaded from orbit.dtu.dk on: Oct 08, 2021 General detection and isolation of specific cells by binding of labeled molecules Pedersen, Henrik; Jakobsen, Søren; Hadrup, Sine Reker; Bentzen, Amalie Kai; Johansen, Kristoffer Haurum Publication date: 2015 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Pedersen, H., Jakobsen, S., Hadrup, S. R., Bentzen, A. K., & Johansen, K. H. (2015). General detection and isolation of specific cells by binding of labeled molecules. (Patent No. WO2015188839). General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/188839 -
Scholarworks@UNO
University of New Orleans ScholarWorks@UNO University of New Orleans Theses and Dissertations Dissertations and Theses Summer 8-4-2011 Identification and characterization of enzymes involved in the biosynthesis of different phycobiliproteins in cyanobacteria Avijit Biswas University of New Orleans, [email protected] Follow this and additional works at: https://scholarworks.uno.edu/td Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Biswas, Avijit, "Identification and characterization of enzymes involved in the biosynthesis of different phycobiliproteins in cyanobacteria" (2011). University of New Orleans Theses and Dissertations. 446. https://scholarworks.uno.edu/td/446 This Dissertation-Restricted is protected by copyright and/or related rights. It has been brought to you by ScholarWorks@UNO with permission from the rights-holder(s). You are free to use this Dissertation-Restricted in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/or on the work itself. This Dissertation-Restricted has been accepted for inclusion in University of New Orleans Theses and Dissertations by an authorized administrator of ScholarWorks@UNO. For more information, please contact [email protected]. Identification and characterization of enzymes involved in biosynthesis of different phycobiliproteins in cyanobacteria A Thesis Submitted to the Graduate Faculty of the University of New Orleans in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Chemistry (Biochemistry) By Avijit Biswas B.S. -
Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology
International Journal of Molecular Sciences Review Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology Jean-Denis Pedelacq 1,* and Stéphanie Cabantous 2,* 1 Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France 2 Centre de Recherche en Cancérologie de Toulouse (CRCT), Inserm, Université Paul Sabatier-Toulouse III, CNRS, 31037 Toulouse, France * Correspondence: [email protected] (J.-D.P.); [email protected] (S.C.) Received: 15 June 2019; Accepted: 8 July 2019; Published: 15 July 2019 Abstract: Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP,that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology. Keywords: fluorescent protein; superfolder; split-GFP; bipartite; tripartite; folding; PPI 1. Superfolder Fluorescent Proteins: Progenitor of Split Fluorescent Protein (FP) Systems Previously described mutations that improve the physical properties and expression of green fluorescent protein (GFP) color variants in the host organism have already been the subject of several reviews [1–4] and will not be described here. -
Intrinsic Indicators for Specimen Degradation
Laboratory Investigation (2013) 93, 242–253 & 2013 USCAP, Inc All rights reserved 0023-6837/13 $32.00 Intrinsic indicators for specimen degradation Jie Li1, Catherine Kil1, Kelly Considine1, Bartosz Smarkucki1, Michael C Stankewich1, Brian Balgley2 and Alexander O Vortmeyer1 Variable degrees of molecular degradation occur in human surgical specimens before clinical examination and severely affect analytical results. We therefore initiated an investigation to identify protein markers for tissue degradation assessment. We exposed 4 cell lines and 64 surgical/autopsy specimens to defined periods of time at room temperature before procurement (experimental cold ischemic time (CIT)-dependent tissue degradation model). Using two-dimen- sional fluorescence difference gel electrophoresis in conjunction with mass spectrometry, we performed comparative proteomic analyses on cells at different CIT exposures and identified proteins with CIT-dependent changes. The results were validated by testing clinical specimens with western blot analysis. We identified 26 proteins that underwent dynamic changes (characterized by continuous quantitative changes, isoelectric changes, and/or proteolytic cleavages) in our degradation model. These changes are strongly associated with the length of CIT. We demonstrate these proteins to represent universal tissue degradation indicators (TDIs) in clinical specimens. We also devised and implemented a unique degradation measure by calculating the quantitative ratio between TDIs’ intact forms and their respective degradation- -
Anti-GFP (Green Fluorescent Protein) Mab-Agarose Code No
D153-8 For Research Use Only. Page 1 of 2 Not for use in diagnostic procedures. MONOCLONAL ANTIBODY Anti-GFP (Green Fluorescent Protein) mAb-Agarose Code No. Clone Subclass Quantity D153-8 RQ2 Rat IgG2a Gel: 200 L BACKGROUND: Since the detection of intracellular 5) Dragone, L. L., et al., PNAS. 103, 18202-18207 (2006) [IP] Aequorea Victria Green Fluorescent Protein (GFP) requires 6) Darzacq, X., et al., J. Cell Biol. 173, 207-218 (2006) [ChIP] only irradiation by UV or blue light, it provides an excellent 7) Hayakawa, T., et al., Plant Cell Physiol. 47, 891-904 (2006) [IP] means for monitoring gene expression and protein 8) Obuse, C., et al., Nat. Cell Biol. 6, 1135-1141 (2004) [IP] localization in living cells. Agarose conjugated anti-GFP monoclonal antibody can detect GFP fusion protein on As this antibody is really famous all over the world, a lot of Immunoprecipitation. researches have been reported. These references are a part of such reports. SOURCE: This antibody was purified from hybridoma (clone RQ2) supernatant using protein G agarose. This INTENDED USE: hybridoma was established by fusion of mouse myeloma For Research Use Only. Not for use in diagnostic procedures. cell PAI with Wister rat lymphnode immunized with GFP purified from GFP expressed 293T cells by affinity 1 2 chromatographic technique using mouse anti-GFP. kDa 66 FORMULATION: 100 g of anti-GFP monoclonal antibody covalently coupled to 200 L of agarose gel and 45 provided as a 50% gel slurry suspended in PBS containing GFP fusion protein preservative (0.1% ProClin 150) for a total volume of 400 30 L. -
Investigations on the Impact of Toxic Cyanobacteria on Fish : As
INVESTIGATIONS ON THE IMPACT OF TOXIC CYANOBACTERIA ON FISH - AS EXEMPLIFIED BY THE COREGONIDS IN LAKE AMMERSEE - DISSERTATION Zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften an der Universität Konstanz Fachbereich Biologie Vorgelegt von BERNHARD ERNST Tag der mündlichen Prüfung: 05. Nov. 2008 Referent: Prof. Dr. Daniel Dietrich Referent: Prof. Dr. Karl-Otto Rothhaupt Referent: Prof. Dr. Alexander Bürkle 2 »Erst seit gestern und nur für einen Tag auf diesem Planeten weilend, können wir nur hoffen, einen Blick auf das Wissen zu erhaschen, das wir vermutlich nie erlangen werden« Horace-Bénédict de Saussure (1740-1799) Pionier der modernen Alpenforschung & Wegbereiter des Alpinismus 3 ZUSAMMENFASSUNG Giftige Cyanobakterien beeinträchtigen Organismen verschiedenster Entwicklungsstufen und trophischer Ebenen. Besonders bedroht sind aquatische Organismen, weil sie von Cyanobakterien sehr vielfältig beeinflussbar sind und ihnen zudem oft nur sehr begrenzt ausweichen können. Zu den toxinreichsten Cyanobakterien gehören Arten der Gattung Planktothrix. Hierzu zählt auch die Burgunderblutalge Planktothrix rubescens, eine Cyanobakterienart die über die letzten Jahrzehnte im Besonderen in den Seen der Voralpenregionen zunehmend an Bedeutung gewonnen hat. An einigen dieser Voralpenseen treten seit dem Erstarken von P. rubescens existenzielle, fischereiwirtschaftliche Probleme auf, die wesentlich auf markante Wachstumseinbrüche bei den Coregonenbeständen (Coregonus sp.; i.e. Renken, Felchen, etc.) zurückzuführen sind. So auch -
Immunohistochemistry Stain Offerings
immunohistochemistry stain offerings TRUSTED PATHOLOGISTS. INVALUABLE ANSWERS.™ MARCHMAY 20172021 www.aruplab.com/ap-ihcaruplab.com/ap-ihc InformationInformation in this brochurein this brochure is current is current as of as May of March 2021. 2017. All content All content is subject is subject to tochange. change. Please contactPlease ARUPcontact ClientARUP Services Client Services at 800-522-2787 at (800) 522-2787 with any with questions any questions or concerns.or concerns. ARUP LABORATORIES As a nonprofit, academic institution of the University of Utah and its Department We believe in of Pathology, ARUP believes in collaborating, sharing and contributing to laboratory science in ways that benefit our clients and their patients. collaborating, Our test menu is one of the broadest in the industry, encompassing more sharing and than 3,000 tests, including highly specialized and esoteric assays. We offer comprehensive testing in the areas of genetics, molecular oncology, pediatrics, contributing pain management, and more. to laboratory ARUP’s clients include many of the nation’s university teaching hospitals and children’s hospitals, as well as multihospital groups, major commercial science in ways laboratories, and group purchasing organizations. We believe that healthcare should be delivered as close to the patient as possible, which is why we support that provide our clients’ efforts to be the principal healthcare provider in the communities they serve by offering highly complex assays and accompanying consultative support. the best value Offering analytics, consulting, and decision support services, ARUP provides for the patient. clients with the utilization management tools necessary to prosper in this time of value-based care. -
Elongation Inhibitors Do Not Prevent the Release Of
RESEARCH ARTICLE Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes Benjamin D Hobson1,2, Linghao Kong1, Erik W Hartwick3, Ruben L Gonzalez3, Peter A Sims1,4,5* 1Department of Systems Biology, Columbia University Irving Medical Center, New York, United States; 2Medical Scientist Training Program, Columbia University Irving Medical Center, New York, United States; 3Department of Chemistry, Columbia University, New York, United States; 4Department of Biochemistry & Molecular Biophysics, Columbia University Irving Medical Center, New York, United States; 5Sulzberger Columbia Genome Center, Columbia University Irving Medical Center, New York, United States Abstract Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti- puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise *For correspondence: subcellular translation sites using anti-puromycin immunostaining may be confounded by release of [email protected] puromycylated nascent polypeptide chains prior to fixation.