Immunohistochemistry Application Guide
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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)}. -
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- -
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. -
Tubulin Or Not Tubulin: Heading Toward Total Protein Staining As Loading Control in Western Blots Christian Moritz
Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots Christian Moritz To cite this version: Christian Moritz. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Proteomics, Wiley-VCH Verlag, 2017, 17 (20), 10.1002/pmic.201600189. hal- 01900776 HAL Id: hal-01900776 https://hal.archives-ouvertes.fr/hal-01900776 Submitted on 22 Oct 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. This is the pre-peer reviewed version of the following article: Moritz, C. P. (2017), Tubulin or not tubulin: Heading towards total protein staining as loading control in Western blots. Proteomics, 1600189. Accepted Author Manuscript. doi:10.1002/pmic.201600189 which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/pmic.201600189/ab stract. If you have access, the final form is recommended as it differs in some parts from the pre-version. This version of the article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. Pre-peer reviewed version of a review article published in PROTEOMICS Tubulin or not tubulin: Heading towards total protein staining as loading control in Western blots. -
Blocking with Immunizing Peptide (Bl) Protocol
BLOCKING WITH IMMUNIZING PEPTIDE (BL) PROTOCOL Non-specific binding of an antibody to proteins other than the antigen can sometimes occur. This is usually more common with polyclonal antibodies, but can also occur with monoclonals as well. To determine which band or staining is specific, an immunizing peptide blocking experiment can be performed. Before proceeding with the staining protocol, the antibody is neutralized (incubated with an excess of peptide that corresponds to the epitope recognized by the antibody). The antibody that is bound to the blocking peptide is no longer available to bind to the epitope present in the protein on the Western blot or in the cell. The neutralized antibody is then used side-by-side with the antibody alone, and the results are compared. By comparing the staining from the blocked antibody versus the antibody alone, you can see which staining is specific: this staining will be absent from the Western blot or immunostaining performed with the neutralized antibody. Materials and Reagents • Blocking buffer (usually TBST plus either 5% non-fat dry milk or 3% BSA for Western blot, or PBS plus 1% BSA for IHC) • Antibody • Blocking (immunizing) peptide • Two tubes • Two identical samples (e.g. a Western blot with two identical lanes, cut in half; two slides containing the cells of interest; etc) Method 1. Determine the optimal concentration of antibody that consistently gives a positive result in your particular protocol. Using that concentration, determine how much antibody you will need for two experiments. a. For example, an antibody is being used successfully in Western blot at 0.5 µg/ml. -
Maccarrone-G.Pdf
Journal of Chromatography B, 1047 (2017) 131–140 Contents lists available at ScienceDirect Journal of Chromatography B jou rnal homepage: www.elsevier.com/locate/chromb MALDI imaging mass spectrometry analysis—A new approach for protein mapping in multiple sclerosis brain lesions a,b,1 a,1 c Giuseppina Maccarrone , Sandra Nischwitz , Sören-Oliver Deininger , a d,e d Joachim Hornung , Fatima Barbara König , Christine Stadelmann , b,1 a,f,∗,1 Christoph W. Turck , Frank Weber a Max Planck Institute of Psychiatry, Kraepelinstr. 2-10, 80804 Munich, Germany b Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Germany c Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany d Institute of Neuropathology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany e Institut für Pathologie, Klinikum Kassel, Mönchebergstr. 41-43, 34125 Kassel, Germany f Medical Park Bad Camberg, Obertorstr. 100-102, 65520 Bad Camberg, Germany a r t i c l e i n f o a b s t r a c t Article history: Multiple sclerosis is a disease of the central nervous system characterized by recurrent inflammatory Received 21 February 2016 demyelinating lesions in the early disease stage. Lesion formation and mechanisms leading to lesion Accepted 1 July 2016 remyelination are not fully understood. Matrix Assisted Laser Desorption Ionisation Mass Spectrom- Available online 1 July 2016 etry imaging (MALDI–IMS) is a technology which analyses proteins and peptides in tissue, preserves their spatial localization, and generates molecular maps within the tissue section. In a pilot study we Keywords: employed MALDI imaging mass spectrometry to profile and identify peptides and proteins expressed in MALDI imaging mass spectrometry normal-appearing white matter, grey matter and multiple sclerosis brain lesions with different extents LC–ESI–MS/MS of remyelination. -
Snomed Ct Dicom Subset of January 2017 Release of Snomed Ct International Edition
SNOMED CT DICOM SUBSET OF JANUARY 2017 RELEASE OF SNOMED CT INTERNATIONAL EDITION EXHIBIT A: SNOMED CT DICOM SUBSET VERSION 1.