Design, Visualize and Detect
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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)}. -
CCR7 Antibody (Y59) NB110-55680
Product Datasheet CCR7 Antibody (Y59) NB110-55680 Unit Size: 0.1 ml Store at -20C. Avoid freeze-thaw cycles. Publications: 9 Protocols, Publications, Related Products, Reviews, Research Tools and Images at: www.novusbio.com/NB110-55680 Updated 5/28/2019 v.20.1 Earn rewards for product reviews and publications. Submit a publication at www.novusbio.com/publications Submit a review at www.novusbio.com/reviews/destination/NB110-55680 Page 1 of 6 v.20.1 Updated 5/28/2019 NB110-55680 CCR7 Antibody (Y59) Product Information Unit Size 0.1 ml Concentration This product is unpurified. The exact concentration of antibody is not quantifiable. Storage Store at -20C. Avoid freeze-thaw cycles. Clonality Monoclonal Clone Y59 Preservative 0.01% Sodium Azide Isotype IgG Purity Tissue culture supernatant Buffer 49% PBS, 0.05% BSA and 50% Glycerol Target Molecular Weight 45 kDa Product Description Host Rabbit Gene ID 1236 Gene Symbol CCR7 Species Human, Mouse, Rat, Monkey Reactivity Notes May cross reacts with Rhesus monkey. Specificity/Sensitivity The antibody does not cross-react with other G-protein coupled receptor 1 family members. Immunogen A synthetic peptide corresponding to residues in N-terminal extracellular domain of human CKR7 was used as immunogen. Notes Produced using Abcam's RabMab® technology. RabMab® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487. Product Application Details Applications Western Blot, Immunocytochemistry/Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry- Paraffin, Immunoprecipitation, Flow Cytometry (Negative) Recommended Dilutions Western Blot 1:1000-10000, Immunohistochemistry 1:10-1:500, Immunocytochemistry/Immunofluorescence 1:250, Immunoprecipitation 1:10, Immunohistochemistry-Paraffin 1:250, Immunohistochemistry-Frozen 1:250, Flow Cytometry (Negative) Application Notes This product is useful for: Western Blot, Immunohistochemistry-Paraffin, Immunocytochemistry, Immunoprecipitation. -
Memoranaurns by the Participants in Signes Par Les Partici- I the Meeting
Memoranda are state- Les Memorandums ments concerning the exposent les conclu- /e , conclusions or recom- sions et recomman- M e mmooranrantedaa mendations of certain dations de certaines / a t w / /WHO scientific meet- /reunions scientifiques ings; they are signed de /'OMS; ils sont Memoranaurns by the participants in signes par les partici- I the meeting. pants a ces reunions. Bulletin ofthe World Health Organization, 62 (2): 217-227 (1984) © World Health Organization 1984 Immunodiagnosis simplified: Memorandum from a WHO Meeting* Technologies suitable for the development ofsimplified immunodiagnostic tests were reviewed by a Working Group of the WHO Advisory Committee on Medical Research in Geneva in June 1983. They included agglutination tests and use ofartificialparticles coated with immunoglobulins, direct visual detection of antigen-antibody reactions, enzyme- immunoassays, and immunofluorescence and fluoroimmunoassays. The use of mono- clonal antibodies,in immunodiagnosis and of DNA /RNA probes to identify viruses was also discussed in detail. The needfor applicability of these tests at three levels, i.e., field conditions (or primary health care level), local laboratories, and central laboratories, was discussed and their use at thefield level was emphasized. Classical serological techniques have been used for All these tests can be carried out in laboratories that a long time for diagnostic purposes, e.g., for con- are equipped with basic instruments as well as special- firmation of clinical diagnoses, epidemiological ized apparatus (e.g., gamma counters for RIA, ultra- studies, testing of blood donors, etc. Some of these violet microscopes for IMF, etc.), which are usually techniques have been standardized to a high degree of available only in the larger, central laboratories. -
Cytokine Immunocytochemistry Fax (65) 860-1590 Fax (49) 40 531 58 92 Fax (81) 3 541-381-55 E-Mail: [email protected]
PharMingen International United States Canada PharMingen Europe Japan PharMingen Canada Tel 619-812-8800 Asia Pacific Becton Dickinson GmbH Nippon Becton Dickinson Toll-Free 1-888-259-0187 Orders 1-800-848-6227 BD Singapore HQ PharMingen Europe Company Ltd. Tel 905-542-8028 Tech Service 1-800-825-5832 Tel (65) 860-1478 Tel (49) 40 532 84 48 0 Tel (81) 3 541-382-51 Fax 905-542-9391 Fax 619-812-8888 Cytokine Immunocytochemistry Fax (65) 860-1590 Fax (49) 40 531 58 92 Fax (81) 3 541-381-55 e-mail: [email protected] http://www.pharmingen.com Africa Germany Latin/South America Spain Becton Dickinson Worldwide Inc. Becton Dickinson GmbH BDIS (USA) Becton Dickinson S.A. Reagents and Techniques Tel (254) 2 449 608 HQ PharMingen Europe Tel (408) 954-2157 Tel (34) 91 848 8100 Fax (254) 2 449 619 Tel (49) 40 532 84 48 0 Fax (408) 526-1804 Fax (34) 91 848 8105 Fax (49) 40 531 58 92 Orders for Microscopic Analysis of Cytokine-Producing Cells Australia Malaysia Tel (34) 91 848 8182 Becton Dickinson Pty Ltd Greece Becton Dickinson Sdn Bhd Fax (34) 91 848 8104 Tel (612) 9978-6800 Becton Dickinson Hellas S.A. Tel (03) 7571323 Fax (612) 9978-6850 Tel (30) 1 9407741 Fax (03) 7571153 Sweden Fax (30) 1 9407740 Becton Dickinson AB Austria Mexico Tel (46) 8 775 51 00 Becton Dickinson GmbH Hong Kong Becton Dickinson de Mexico Fax (46) 8 645 08 08 HQ PharMingen Europe Becton Dickinson Asia Ltd Tel (52-5) 237-12-98 Tel (49) 40 532 84 48 0 Tel (852) 2572-8668 Fax (52-5) 237-12-93 Switzerland Fax (49) 40 531 58 92 Fax (852) 2520-1837 Becton Dickinson GmbH Middle East HQ PharMingen Europe Belgium Hungary Becton Dickinson Tel (41) 06 1-385 4422 Becton Dickinson Benelux N.V. -
Red Blood Cell Preparation and Hemagglutination Assay
Red Blood Cell Preparation and Hemagglutination Assay Purpose: This protocol describes the preparation of RBCs for storage and use in Hemagluttination assays, which are used to determine the La Sota B1 lentogenic Newcastle Disease virion hemaglutinin-neuraminidase titer relative to virion stock positive controls. The protocol was adapted from McGinnes et al. (2006) and “Detection of Hemagglutinating Viruses” (n.d.). Materials: ● Whole Blood Cells ● 50mL conical tube ● Multichannel micropipette, micropipette, and tips ● PBS with 2mM Penicillin/Streptomycin ● SV3 ● Alsever’s Solution ● 96 round bottom well plate ● Microscope slide ● Virion stock ● Centrifuge Procedure: 1. RBC preparation a. Obtain 25mL whole blood and add 25mL cold Alsever’s Solution for anti-coagulation in a 50mL conical tube and keep on ice b. Centrifuge whole blood at 500 RCF for 10min c. Aspirate blood plasma, buffy layer, and top erythrocytes d. Wash 3 times by resuspending in PBS with 2mM Penicillin/Streptomycin to double the pellet volume, centrifuging at 500 RCF, and aspirating the supernatant e. Resuspend the pellet in PBS with 2mM Penicillin/Streptomycin or SV3 for a final concentration of 10% pellet volume per total volume, and store at 2-7˚C for 1 week or 42 days 2. HA Titer a. Thaw your virion stock and samples on ice b. Use a multichannel micropipette to add 50µL of cold PBS to each row on a 96 round bottom well plate for each sample in duplicates c. Add 50µL of each sample to column 1 of their respective duplicate rows i. Don’t add anything to the negative control rows and add virion stocks to the positive control rows d. -
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. -
The Nuclear Membrane Determines the Timing of DNA Replication in Xenopus Egg Extracts Gregory H
The Nuclear Membrane Determines the Timing of DNA Replication in Xenopus Egg Extracts Gregory H. Leno and Ronald A. Laskey Cancer Research Campaign Molecular Embryology Group, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, England Abstract. We have exploited a property of chicken scribed in cultured cells. In contrast to the asynchro- erythrocyte nuclei to analyze the regulation of DNA nous replication seen :between individual nuclei, repli- replication in a cell-free system from Xenopus eggs. cation within multinuclear aggregates was syn- Many individual demembranated nuclei added to the chronous. There ;was a uniform distribution and extract often became enclosed within a common nu- similar fluorescent intensity of the replication loci clear membrane. Nuclei within such a "multinuclear throughout all the nuclei enclosed within the same aggregate" lacked individual membranes but shared the membrane. However, different multinuclear aggregates perimeter membrane of the aggregate. Individual replicated out of synchrony with each other indicating nuclei that were excluded from the aggregates initiated that each membrane-bound aggregate acts as an in- DNA synthesis at different times over a 10-12-h dividual unit of replication. These data indicate that period, as judged by incorporation of biotinylated the nuclear membrane defines the unit of DNA repli- dUTP into discrete replication foci at early times, fol- cation and determines the timing of DNA synthesis in lowed by uniformly intense incorporation at later egg extract resulting in highly coordinated triggering times. Replication forks were clustered in spots, rings, of DNA replication on the DNA it encloses. and horsesh0e-shaped structures similar to those de- NITIATION of DNA replication occurs at thousands of defines the unit of DNA replication in the egg extract. -
AG 39: Immunofluorescence Assays (PDF)
ibidi Application Guide Immunofluorescence Assays The Principle of Immunofluorescence Immunofluorescence Applied: Assays . 2 Experimental Examples . 13 Rat Hippocampal Neuron and Astrocyte Staining 14 Immunofluorescence Staining: Visualization of Endothelial Cell Junctions . 13 A Typical Workflow . 3 Immunostaining of Rat Dorsal Root Ganglionic Experiment Planning and Sample Preparation . 4 Cells and Schwann Cells . 13 Sample Fixation . 4 Adherens Junctions and Actin Cytoskeleton of Cell Permeabilization . 5 HUVECs Under Flow . 14 Blocking . 5 Mitochondria Staining of MDCK cells . 14 Primary Antibody Incubation . 5 Focal Adhesions of Differentiated Mouse Fibroblasts on an Elastic Surface . 15 Secondary Antibody Incubation . 6 Counterstain and Mounting . 7 Microscopy . 7 Troubleshooting . 8 Selected Publications Immunofluorescence C. Xu, et al. NPTX2 promotes colorectal cancer growth and liver With the ibidi Chambers . 9 metastasis by the activation of the canonical Wnt/beta-catenin pathway via FZD6. Cell Death & Disease, 2019, 10.1038/s41419- Comparison of Immunocytochemistry Protocols . 10 019-1467-7 Chambered Coverslips . 11 read abstract Kobayashi, T., et al. Principles of early human development and Channel Slides . 11 germ cell program from conserved model systems. Nature, Chamber Slides . 12 2017, 10.1038/nature22812 read abstract H. Tada et al. Porphyromonas gingivalis Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells. PloS one, 2016, 10.1371/journal.pone.0152794 read abstract N. J. Foy, M. Akhrymuk, A. V. Shustov, E. I. Frolova and I. Frolov. Hypervariable Domain of Nonstructural Protein nsP3 of Venezuelan Equine Encephalitis Virus Determines Cell-Specific Mode of Virus Replication. Journal of Virology, 2013, 10.1128/ jvi.00720-13 read abstract .com The Principle of Immunofluorescence Assays Immunofluorescence (IF) is a powerful approach for getting insight into cellular structures and processes using microscopy . -
Ab228551 Hoechst 33342 Staining Dye Solution
Version 1 Last updated 12 March 2018 ab228551 Hoechst 33342 Staining Dye Solution For labeling DNA in fluorescence microscopy. This product is for research use only and is not intended for diagnostic use. Copyright © 2017 Abcam. All rights reserved Table of Contents 1. Overview 1 2. Materials Supplied and Storage 3 3. Materials Required, Not Supplied 4 4. General guidelines, precautions, and troubleshooting 5 5. Reagent Preparation 6 6. Assay Procedure 7 7. FAQs / Troubleshooting 8 8. Notes 9 Copyright © 2017 Abcam. All rights reserved 1. Overview Hoechst 33342 Staining Dye Solution (ab228551) is a fluorescent stain for labeling DNA in fluorescence microscopy. This product may be used in fluorescence microscopy, microplate, cuvette, and flow cytometry applications. It can also be used to detect the contents of a sample DNA by plotting a standard emission-to content curve. Figure 1. Chemical structure of Hoechst 33342. Figure 2. Spectrum of Hoechst 33342. ab228551 Hoechst 33342 Staining Dye Solution 1 Pellet cells by centrifugation. Resuspend the cells in buffered salt solutions or media, with optimal dye binding at pH 7.4. Add Hoechst stain using concentrations between 0.5 and 5 µM and incubate for 15 to 60 minutes. ab228551 Hoechst 33342 Staining Dye Solution 2 2. Materials Supplied and Storage Store kit at -20°C in the dark immediately on receipt and check below for storage for individual components. Kit can be stored for 1 year from receipt, if components have not been reconstituted. Storage temperatur Item Quantity e (before prep) Hoechst 33342 5 mL -20°C (20 mM Solution in Water) ab228551 Hoechst 33342 Staining Dye Solution 3 3. -
Radioimmunoassay in Developing Countries: General Principles
XA9847613 Chapter 16 RADIOIMMUNOASSAY IN DEVELOPING COUNTRIES (General principles) R.D. Piyasena Radioimmunoassay (RIA) is probably the most commonly performed nuclear medicine technique. It is an in vitro procedure, where no radioactivity is administered to the patient. But this alone is not the reason for its widespread use. It provides the basis for extremely sensitive and specific diagnostic tests, and its use in present day medicine has brought a virtual information explosion in terms of understanding the pathophysiology of many diseases. The fact that the technology involved is within the technical and economic capabilities of the developing world is evident from the increasing demand for its introduction or expansion of existing services. RIA facilities need not be restricted to urban hospitals, as in the case of in vivo nuclear medicine techniques, but may be extended to smaller district hospitals and other laboratories in peripheral areas. It is also possible to send blood samples to a central laboratory so that a single centre can serve a wide geographical area. There are many laboratories in the industrialized world that receive a major proportion of samples for assay by mail. In recent years, substantial RIA services have been established in many of the developing countries in Asia and Latin America. The International Atomic Energy Agency (IAEA) and World Health Organisations (WHO) have made vital contributions to these activities and have played a catalytic role in assisting member states to achieve realistic goals. In the past five years, more than 250 individual RIA laboratories in developing member states have been beneficiaries of IAEA projects.