Live visualization of protein synthesis in axonal growth cones by microinjection of photoconvertible Kaede into Xenopus embryos.

Abstract:

:Photoconvertible fluorescent proteins, such as Kaede, can be switched irreversibly from their native color to a new one. This property can be exploited to visualize de novo mRNA translation, because newly synthesized proteins can be distinguished from preexisting ones by their color. In this protocol, Kaede cDNA linked to the 3' untranslated region (UTR) of beta-actin is delivered into cells fated to become the retina by injection into Xenopus blastomeres. Brief exposure (6-10 s) to UV light (350-410 nm) of Kaede-positive retinal axons/growth cones efficiently converts Kaede from its native green fluorescence to red. The reappearance of the green signal reports the synthesis of new Kaede protein. This approach can be used to investigate the spatiotemporal control of translation of specific mRNAs in response to external stimuli and to test the efficiency of full-length versus mutant UTRs. The 3-d protocol can be adapted for broad use with other photoactivatable fluorescent proteins.

journal_name

Nat Protoc

journal_title

Nature protocols

authors

Leung KM,Holt CE

doi

10.1038/nprot.2008.113

subject

Has Abstract

pub_date

2008-01-01 00:00:00

pages

1318-27

issue

8

eissn

1754-2189

issn

1750-2799

pii

nprot.2008.113

journal_volume

3

pub_type

杂志文章
  • Synthesis of an ultrasensitive BODIPY-derived fluorescent probe for detecting HOCl in live cells.

    abstract::Hypochlorous acid (HOCl) is a critical member of the reactive oxygen species (ROS) produced by immune cells to fight infections. On the other hand, HOCl in homeostasis causes oxidative damage to biomolecules and is linked to many diseases, including inflammatory, neurodegenerative, and cardiovascular diseases. Herein,...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/s41596-018-0041-6

    authors: Zhu H,Zhang Z,Long S,Du J,Fan J,Peng X

    更新日期:2018-10-01 00:00:00

  • In vivo single-cell electroporation for transfer of DNA and macromolecules.

    abstract::Single-cell electroporation allows transfection of plasmid DNA or macromolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and comb...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2006.186

    authors: Bestman JE,Ewald RC,Chiu SL,Cline HT

    更新日期:2006-01-01 00:00:00

  • Genetically modified mouse models to help fight COVID-19.

    abstract::The research community is in a race to understand the molecular mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, to repurpose currently available antiviral drugs and to develop new therapies and vaccines against coronavirus disease 2019 (COVID-19). One major challenge in achieving ...

    journal_title:Nature protocols

    pub_type: 杂志文章,评审

    doi:10.1038/s41596-020-00403-2

    authors: Gurumurthy CB,Quadros RM,Richardson GP,Poluektova LY,Mansour SL,Ohtsuka M

    更新日期:2020-12-01 00:00:00

  • Ex vivo perfusion of mid-to-late-gestation mouse placenta for maternal-fetal interaction studies during pregnancy.

    abstract::Ex vivo perfusion systems offer a reliable, reproducible method for studying acute physiological responses of an organ to various environmental manipulations. Unlike in vitro culture systems, the cellular organization, compartmentalization and three-dimensional structure of ex vivo-perfused organs are maintained. Thes...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2012.144

    authors: Goeden N,Bonnin A

    更新日期:2013-01-01 00:00:00

  • Housing conditions and stimulus females: a robust social discrimination task for studying male rodent social recognition.

    abstract::Social recognition (SR) enables rodents to distinguish between familiar and novel conspecifics, largely through individual odor cues. SR tasks utilize the tendency for a male to sniff and interact with a novel individual more than a familiar individual. Many paradigms have been used to study the roles of the neuropept...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2009.141

    authors: Macbeth AH,Edds JS,Young WS 3rd

    更新日期:2009-01-01 00:00:00

  • Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism.

    abstract::Accurate analysis of DNA sequence variation in not only humans and animals but also other organisms has played a significant role in expanding our knowledge about genetic variety and diversity in a number of different biological areas. The search for an understanding of the causes of genetic variants and mutations has...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2007.407

    authors: Ota M,Fukushima H,Kulski JK,Inoko H

    更新日期:2007-01-01 00:00:00

  • Coupling surface plasmon resonance to mass spectrometry to discover novel protein-protein interactions.

    abstract::The elucidation of protein-protein interaction networks is a crucial task in the postgenomic era. In this protocol, we describe our approach to discover protein-protein interactions using the surface plasmon resonance technique coupled to mass spectrometry (MS). A peptide or a protein is immobilized on a sensor chip a...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2009.84

    authors: Madeira A,Ohman E,Nilsson A,Sjögren B,Andrén PE,Svenningsson P

    更新日期:2009-01-01 00:00:00

  • Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo.

    abstract::A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L27...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/s41596-018-0106-6

    authors: Alvarez-Castelao B,Schanzenbächer CT,Langer JD,Schuman EM

    更新日期:2019-02-01 00:00:00

  • Synthesis of complex head-to-side-chain cyclodepsipeptides.

    abstract::Cyclodepsipeptides are cyclic peptides in which at least one amide link on the backbone is replaced with an ester link. These natural products present a high structural diversity that corresponds to a broad range of biological activities. Therefore, they are very promising pharmaceutical candidates. Most of the cyclod...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2016.116

    authors: Pelay-Gimeno M,Albericio F,Tulla-Puche J

    更新日期:2016-10-01 00:00:00

  • Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing.

    abstract::The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has bec...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2011.396

    authors: Harakalova M,Mokry M,Hrdlickova B,Renkens I,Duran K,van Roekel H,Lansu N,van Roosmalen M,de Bruijn E,Nijman IJ,Kloosterman WP,Cuppen E

    更新日期:2011-11-03 00:00:00

  • High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    abstract::AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and e...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2015.044

    authors: Yu X,LaBaer J

    更新日期:2015-05-01 00:00:00

  • Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol.

    abstract::The telomeric repeat amplification protocol (TRAP) is a two-step process for analyzing telomerase activity in cell or tissue extracts. Recent modifications of this sensitive assay include elimination of radioactivity by using a fluorescently labeled primer instead of a radiolabeled primer. In addition, the TRAP assay ...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2006.239

    authors: Herbert BS,Hochreiter AE,Wright WE,Shay JW

    更新日期:2006-01-01 00:00:00

  • Fabrication of carbohydrate chips and their use to probe protein-carbohydrate interactions.

    abstract::Carbohydrate microarrays have received considerable attention as an advanced technology for the rapid analysis of carbohydrate-protein interactions. This protocol provides detailed procedures for the preparation of carbohydrate microarrays by immobilizing hydrazide-conjugated carbohydrates on epoxide-derivatized glass...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2007.373

    authors: Park S,Lee MR,Shin I

    更新日期:2007-01-01 00:00:00

  • Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells.

    abstract::Forebrain γ-aminobutyric acid (GABA) interneurons have crucial roles in high-order brain function via modulating network activities and plasticity, and they are implicated in many psychiatric disorders. Availability of enriched functional human forebrain GABA interneurons, especially those from people affected by GABA...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2013.106

    authors: Liu Y,Liu H,Sauvey C,Yao L,Zarnowska ED,Zhang SC

    更新日期:2013-09-01 00:00:00

  • A convenient, high-throughput method for enzyme-luminescence detection of dopamine released from PC12 cells.

    abstract::This protocol represents a novel enzyme-luminescence method to detect dopamine sensitively and rapidly with high temporal resolution. In principle, dopamine is first oxidized with tyramine oxidase to produce H(2)O(2), and then the produced H(2)O(2) reacts with luminol to generate chemiluminescence in the presence of h...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2008.158

    authors: Shinohara H,Wang F,Hossain SM

    更新日期:2008-01-01 00:00:00

  • Application of cationic conjugated polymers in microarrays using label-free DNA targets.

    abstract::A fluorescence-based microarray technique that does not require target DNA labeling is detailed. This 'label-free' approach utilizes a cationic, water-soluble conjugated polymer PFBT (poly[9,9'-bis(6''-(N,N,N-trimethylammonium)hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole) dibromide]), and neutral PNA (peptide nuc...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2007.307

    authors: Sun C,Gaylord BS,Hong JW,Liu B,Bazan GC

    更新日期:2007-01-01 00:00:00

  • Post-translational mutagenesis for installation of natural and unnatural amino acid side chains into recombinant proteins.

    abstract::Methods for installing natural and unnatural amino acids and their modifications into proteins in a benign and precise manner are highly sought-after in protein science. Here we describe a protocol for 'post-translational mutagenesis' that enables the programmed installation of protein side chains through the use of r...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2017.087

    authors: Wright TH,Davis BG

    更新日期:2017-10-01 00:00:00

  • Synthesis and application of an azobenzene amino acid as a light-switchable turn element in polypeptides.

    abstract::The synthesis of an azobenzene amino acid (aa) for use as a photo-inducible conformational switch in polypeptides is described. The compound can be easily incorporated into an aa sequence by solid-phase peptide synthesis using standard 9-fluorenylmethoxycarbonyl methods. A reversible conformational change of the pepti...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2006.488

    authors: Aemissegger A,Hilvert D

    更新日期:2007-01-01 00:00:00

  • Determining the polysaccharide composition of plant cell walls.

    abstract::The plant cell wall is a chemically complex structure composed mostly of polysaccharides. Detailed analyses of these cell wall polysaccharides are essential for our understanding of plant development and for our use of plant biomass (largely wall material) in the food, agriculture, fabric, timber, biofuel and biocompo...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2012.081

    authors: Pettolino FA,Walsh C,Fincher GB,Bacic A

    更新日期:2012-09-01 00:00:00

  • Silver staining DNA in polyacrylamide gels.

    abstract::This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described pro...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2007.330

    authors: Bassam BJ,Gresshoff PM

    更新日期:2007-01-01 00:00:00

  • Reconstitution of the cell cycle-regulated Golgi disassembly and reassembly in a cell-free system.

    abstract::The Golgi apparatus undergoes extensive disassembly during mitosis and reassembly in post-mitotic daughter cells. This process has been mimicked in vitro by treating Golgi membranes with mitotic and interphase cytosol. To determine the minimal machinery that controls the morphological change, we have developed a defin...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2010.38

    authors: Tang D,Xiang Y,Wang Y

    更新日期:2010-04-01 00:00:00

  • Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels.

    abstract::This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli c...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2012.051

    authors: Shin Y,Han S,Jeon JS,Yamamoto K,Zervantonakis IK,Sudo R,Kamm RD,Chung S

    更新日期:2012-06-07 00:00:00

  • Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons.

    abstract::The pig has emerged as an important large animal model in biomedical and pharmaceutical research. We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection of a plasmid encoding ...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2014.010

    authors: Ivics Z,Garrels W,Mátés L,Yau TY,Bashir S,Zidek V,Landa V,Geurts A,Pravenec M,Rülicke T,Kues WA,Izsvák Z

    更新日期:2014-04-01 00:00:00

  • Self-assembly and characterization of 2D plasmene nanosheets.

    abstract::Freestanding plasmonic nanoparticle (NP) superlattice sheets are novel 2D nanomaterials with tailorable properties that enable their use for broad applications in sensing, anticounterfeit measures, ionic gating, nanophotonics and flat lenses. We recently developed a robust, yet general, two-step drying-mediated approa...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/s41596-019-0200-4

    authors: Dong D,Fu R,Shi Q,Cheng W

    更新日期:2019-09-01 00:00:00

  • A general pipeline for quality and statistical assessment of protein interaction data using R and Bioconductor.

    abstract::The systematic mapping of protein interactions by bait-prey techniques, including affinity purification-mass spectrometry or the yeast two-hybrid system, contributes a unique and relevant perspective on the comprehensive picture of cellular machines. We describe here a protocol for statistical analysis of node-and-edg...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2009.26

    authors: Chiang T,Scholtens D

    更新日期:2009-01-01 00:00:00

  • Generation of a transgenic ORFeome library in Drosophila.

    abstract::Overexpression screens can be used to explore gene function in Drosophila melanogaster, but to demonstrate their full potential, comprehensive and systematic collections of fly strains are required. Here we provide a protocol for high-throughput cloning of Drosophila open-reading frames (ORFs) that are regulated by up...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2014.105

    authors: Bischof J,Sheils EM,Björklund M,Basler K

    更新日期:2014-07-01 00:00:00

  • Buccal micronucleus cytome assay.

    abstract::The Buccal Micronucleus Cytome (BMCyt) assay is a minimally invasive method for studying DNA damage, chromosomal instability, cell death and the regenerative potential of human buccal mucosal tissue. This method is increasingly used in molecular epidemiological studies for investigating the impact of nutrition, lifest...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2009.53

    authors: Thomas P,Holland N,Bolognesi C,Kirsch-Volders M,Bonassi S,Zeiger E,Knasmueller S,Fenech M

    更新日期:2009-01-01 00:00:00

  • A practical guide to adaptive light-sheet microscopy.

    abstract::We describe the implementation and use of an adaptive imaging framework for optimizing spatial resolution and signal strength in a light-sheet microscope. The framework, termed AutoPilot, comprises hardware and software modules for automatically measuring and compensating for mismatches between light-sheet and detecti...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/s41596-018-0043-4

    authors: Royer LA,Lemon WC,Chhetri RK,Keller PJ

    更新日期:2018-11-01 00:00:00

  • Design of a functional cyclic HSV1-TK reporter and its application to PET imaging of apoptosis.

    abstract::Positron emission tomography (PET) is a sensitive and noninvasive imaging method that is widely used to explore molecular events in living subjects. PET can precisely and quantitatively evaluate cellular apoptosis, which has a crucial role in various physiological and pathological processes. In this protocol, we descr...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2015.048

    authors: Wang Z,Wang F,Hida N,Kiesewetter DO,Tian J,Niu G,Chen X

    更新日期:2015-05-01 00:00:00

  • Synthesis of alkyl- and aryl-amino-substituted anthraquinone derivatives by microwave-assisted copper(0)-catalyzed Ullmann coupling reactions.

    abstract::This protocol describes the efficient, generally applicable Ullmann coupling reaction of bromaminic acid with alkyl- or aryl-amines in phosphate buffer under microwave irradiation using elemental copper as a catalyst. The reaction leads to a number of biologically active compounds. As a prototypical example, the synth...

    journal_title:Nature protocols

    pub_type: 杂志文章

    doi:10.1038/nprot.2010.63

    authors: Baqi Y,Müller CE

    更新日期:2010-05-01 00:00:00