Transient expression and analysis of fluorescent reporter proteins in plant pollen tubes.


:The pollen tube is an excellent single-cell model system for studying cellular processes in plant cell biology. This protocol describes a detailed step-by-step procedure with optimized conditions for introducing various fluorescent reporter proteins into lily, tobacco and Arabidopsis pollen grains by means of biolistics for their transient expression and subsequent analysis in germinating pollen tubes. The whole experiment consists of four major stages: coating gold microcarriers with DNA constructs, preparation of pollen grains, transformation of plasmid DNA into pollen grains by particle delivery system and germination of bombarded pollen grains in optimized germination media to obtain pollen tubes for protein trafficking, protein localization, drug treatment and organelle dynamics analysis. This protocol takes about 4-12 h from pollen preparation to protein detection.


Nat Protoc


Nature protocols


Wang H,Jiang L




Has Abstract


2011-04-01 00:00:00














  • Synthesis of enantiomerically enriched (R)-5-tert-butylazepan-2-one using a hydroxyalkyl azide mediated ring-expansion reaction.

    abstract::A procedure for the conversion of a symmetrical ketone to an enantiomerically pure lactam is described. The technique described here involves a ring-expansion reaction of a 4-substituted cyclohexanone accomplished with a chiral 1,3-azidopropanol derivative. The procedure entails first a one-step preparation of (R)-1-p...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Ribelin TP,Aubé J

    更新日期:2008-01-01 00:00:00

  • Recombineering: a homologous recombination-based method of genetic engineering.

    abstract::Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in Escherichia coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraint...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Sharan SK,Thomason LC,Kuznetsov SG,Court DL

    更新日期:2009-01-01 00:00:00

  • An experimental murine model to study periodontitis.

    abstract::Periodontal disease (PD) is a common dental disease associated with the interaction between dysbiotic oral microbiota and host immunity. It is a prevalent disease, resulting in loss of gingival tissue, periodontal ligament, cementum and alveolar bone. PD is a major form of tooth loss in the adult population. Experimen...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Marchesan J,Girnary MS,Jing L,Miao MZ,Zhang S,Sun L,Morelli T,Schoenfisch MH,Inohara N,Offenbacher S,Jiao Y

    更新日期:2018-10-01 00:00:00

  • Holographic two-photon activation for synthetic optogenetics.

    abstract::Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammal...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Carmi I,De Battista M,Maddalena L,Carroll EC,Kienzler MA,Berlin S

    更新日期:2019-03-01 00:00:00

  • Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates.

    abstract::Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the a...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Tanase M,Zolla V,Clement CC,Borghi F,Urbanska AM,Rodriguez-Navarro JA,Roda B,Zattoni A,Reschiglian P,Cuervo AM,Santambrogio L

    更新日期:2015-01-01 00:00:00

  • Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method.

    abstract::Here we describe a protocol for the production of frozen competent yeast cells that can be transformed with high efficiency using the lithium acetate/single-stranded carrier DNA/PEG method. This protocol allows the production of highly competent yeast cells that can be frozen and used at a later date and is especially...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Gietz RD,Schiestl RH

    更新日期:2007-01-01 00:00:00

  • 3D mouse embryonic stem cell culture for generating inner ear organoids.

    abstract::This protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 9...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Koehler KR,Hashino E

    更新日期:2014-01-01 00:00:00

  • A general purpose method for extracting RNA from Dictyostelium cells.

    abstract::Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba that undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Pilcher KE,Gaudet P,Fey P,Kowal AS,Chisholm RL

    更新日期:2007-01-01 00:00:00

  • Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing.

    abstract::Gene tagging with fluorescent proteins is essential for investigations of the dynamic properties of cellular proteins. CRISPR-Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and allows functionality and physiological expression of the fusion protein. ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Koch B,Nijmeijer B,Kueblbeck M,Cai Y,Walther N,Ellenberg J

    更新日期:2018-06-01 00:00:00

  • Long term non-invasive imaging of embryonic stem cells using reporter genes.

    abstract::Development of non-invasive and accurate methods to track cell fate after delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. In this protocol, we describe the in vivo monitoring of stem cell survival, proliferation and migration using reporter genes. We established stable ES c...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Sun N,Lee A,Wu JC

    更新日期:2009-01-01 00:00:00

  • Site-specific protein labeling using PRIME and chelation-assisted click chemistry.

    abstract::This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Uttamapinant C,Sanchez MI,Liu DS,Yao JZ,Ting AY

    更新日期:2013-08-01 00:00:00

  • Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt.

    abstract::This protocol describes procedures for performing fluorescence resonance energy transfer (FRET) microscopy analysis by three different methods: acceptor photobleaching, sensitized emission and spectral imaging. We also discuss anisotropy and fluorescence lifetime imaging microscopy-based FRET techniques. By using the ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Broussard JA,Rappaz B,Webb DJ,Brown CM

    更新日期:2013-02-01 00:00:00

  • Isolation of DNA from small amounts of elephant ivory.

    abstract::This protocol describes a method for the extraction of DNA from elephant ivory. These techniques are being used to assign geographic origin to poached ivory by comparing the ivory genotype to a geographic-based gene frequency map, developed separately. The method has three components: ivory pulverization, decalcificat...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Mailand C,Wasser SK

    更新日期:2007-01-01 00:00:00

  • Characterizing peptides in individual mammalian cells using mass spectrometry.

    abstract::Cell-to-cell chemical signaling plays multiple roles in coordinating the activity of the functional elements of an organism, with these elements ranging from a three-neuron reflex circuit to the entire animal. In recent years, single-cell mass spectrometry (MS) has enabled the discovery of cell-to-cell signaling molec...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Rubakhin SS,Sweedler JV

    更新日期:2007-01-01 00:00:00

  • Using Raman spectroscopy to characterize biological materials.

    abstract::Raman spectroscopy can be used to measure the chemical composition of a sample, which can in turn be used to extract biological information. Many materials have characteristic Raman spectra, which means that Raman spectroscopy has proven to be an effective analytical approach in geology, semiconductor, materials and p...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Butler HJ,Ashton L,Bird B,Cinque G,Curtis K,Dorney J,Esmonde-White K,Fullwood NJ,Gardner B,Martin-Hirsch PL,Walsh MJ,McAinsh MR,Stone N,Martin FL

    更新日期:2016-04-01 00:00:00

  • Genome editing comes of age.

    abstract::Genome editing harnesses programmable nucleases to cut and paste genetic information in a targeted manner in living cells and organisms. Here, I review the development of programmable nucleases, including zinc finger nucleases (ZFNs), TAL (transcription-activator-like) effector nucleases (TALENs) and CRISPR (cluster o...

    journal_title:Nature protocols

    pub_type: 杂志文章,评审


    authors: Kim JS

    更新日期:2016-09-01 00:00:00

  • Synthesis of [(18)F]SiFB: a prosthetic group for direct protein radiolabeling for application in positron emission tomography.

    abstract::N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB) is a highly reactive prosthetic group for radiolabeling of proteins for use in positron emission tomography (PET). It is similar to N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB), the 'gold-standard' prosthetic group for protein (18)F-labeli...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Kostikov AP,Chin J,Orchowski K,Schirrmacher E,Niedermoser S,Jurkschat K,Iovkova-Berends L,Wängler C,Wängler B,Schirrmacher R

    更新日期:2012-11-01 00:00:00

  • High spatial resolution imaging of biological tissues using nanospray desorption electrospray ionization mass spectrometry.

    abstract::Mass spectrometry imaging (MSI) enables label-free spatial mapping of hundreds of biomolecules in tissue sections. This capability provides valuable information on tissue heterogeneity that is difficult to obtain using population-averaged assays. Despite substantial developments in both instrumentation and methodology...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Yin R,Burnum-Johnson KE,Sun X,Dey SK,Laskin J

    更新日期:2019-12-01 00:00:00

  • A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation.

    abstract::The specialized microfold cells (M cells) in the follicle-associated epithelium (FAE) of intestinal Peyer's patches serve as antigen-sampling cells of the intestinal innate immune system. Unlike 'classical' enterocytes, they are able to translocate diverse particulates without digesting them. They act as pathways for ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Beloqui A,Brayden DJ,Artursson P,Préat V,des Rieux A

    更新日期:2017-07-01 00:00:00

  • Using hyperLOPIT to perform high-resolution mapping of the spatial proteome.

    abstract::The organization of eukaryotic cells into distinct subcompartments is vital for all functional processes, and aberrant protein localization is a hallmark of many diseases. Microscopy methods, although powerful, are usually low-throughput and dependent on the availability of fluorescent fusion proteins or highly specif...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Mulvey CM,Breckels LM,Geladaki A,Britovšek NK,Nightingale DJH,Christoforou A,Elzek M,Deery MJ,Gatto L,Lilley KS

    更新日期:2017-06-01 00:00:00

  • A whole-tissue RNA-seq toolkit for organism-wide studies of gene expression with PME-seq.

    abstract::The immune system operates at the scale of the whole organism in mammals. We currently lack experimental approaches to systematically track and study organism-wide molecular processes in mice. Here we describe an integrated toolkit for measuring gene expression in whole tissues, 3-prime mRNA extension sequencing, that...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Pandey S,Takahama M,Gruenbaum A,Zewde M,Cheronis K,Chevrier N

    更新日期:2020-04-01 00:00:00

  • Data analysis of assorted serum peptidome profiles.

    abstract::Discovery of biomarker patterns using proteomic techniques requires examination of large numbers of patient and control samples, followed by data mining of the molecular read-outs (e.g., mass spectra). Adequate signal processing and statistical analysis are critical for successful extraction of markers from these data...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Villanueva J,Philip J,DeNoyer L,Tempst P

    更新日期:2007-01-01 00:00:00

  • Construction of BIBAC and BAC libraries from a variety of organisms for advanced genomics research.

    abstract::Large-insert BAC (bacterial artificial chromosome) and BIBAC (binary BAC) libraries are essential for modern genomics research for all organisms. We helped pioneer the BAC and BIBAC technologies, and by using them we have constructed hundreds of BAC and BIBAC libraries for different species of plants, animals, marine ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Zhang HB,Scheuring CF,Zhang M,Zhang Y,Wu CC,Dong JJ,Li Y

    更新日期:2012-02-16 00:00:00

  • Direct metabolomics for plant cells by live single-cell mass spectrometry.

    abstract::Live single-cell mass spectrometry (live MS) provides a mass spectrum that shows thousands of metabolite peaks from a single live plant cell within minutes. By using an optical microscope, a cell is chosen for analysis and a metal-coated nanospray microcapillary tip is used to remove the cell's contents. After adding ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Fujii T,Matsuda S,Tejedor ML,Esaki T,Sakane I,Mizuno H,Tsuyama N,Masujima T

    更新日期:2015-09-01 00:00:00

  • Video tracking and analysis of sleep in Drosophila melanogaster.

    abstract::In the past decade, Drosophila has emerged as an ideal model organism for studying the genetic components of sleep as well as its regulation and functions. In fruit flies, sleep can be conveniently estimated by measuring the locomotor activity of the flies using techniques and instruments adapted from the field of cir...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Gilestro GF

    更新日期:2012-04-26 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


    authors: Aemissegger A,Hilvert D

    更新日期:2007-01-01 00:00:00

  • Single-molecule mRNA detection and counting in mammalian tissue.

    abstract::We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecul...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Lyubimova A,Itzkovitz S,Junker JP,Fan ZP,Wu X,van Oudenaarden A

    更新日期:2013-09-01 00:00:00

  • Vertebrate neural stem cell segmentation, tracking and lineaging with validation and editing.

    abstract::This protocol and the accompanying software program called LEVER (lineage editing and validation) enable quantitative automated analysis of phase-contrast time-lapse images of cultured neural stem cells. Images are captured at 5-min intervals over a period of 5-15 d as the cells proliferate and differentiate. LEVER au...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Winter M,Wait E,Roysam B,Goderie SK,Ali RA,Kokovay E,Temple S,Cohen AR

    更新日期:2011-11-17 00:00:00

  • Standardized bacteriophage purification for personalized phage therapy.

    abstract::The world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward-by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Luong T,Salabarria AC,Edwards RA,Roach DR

    更新日期:2020-09-01 00:00:00

  • Selective chemoprecipitation to enrich nitropeptides from complex proteomes for mass-spectrometric analysis.

    abstract::Post-translational protein nitration has attracted interest owing to its involvement in cellular signaling, effects on protein function and potential as biomarker of nitroxidative stress. We describe a procedure for enriching nitropeptides for mass spectrometry (MS)-based proteomics that is a simple and reliable alter...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Prokai L,Guo J,Prokai-Tatrai K

    更新日期:2014-04-01 00:00:00