Name: Isolamento e purificação de vesículas extracelulares vegetais de folhas de Arabidopsis usando um método otimizado de coleta por lavagem apoplástica (Vídeo) | JoVE Journal
Uploaded: 2026-03-24T12:50:00.000+00:00
Duration: 9 min 38 s
Description: Aprenda a isolar vesículas extracelulares de folhas de Arabidopsis thaliana usando infiltração suave e gradiente de sacarose. Este protocolo minimiza danos celulares e contaminação citoplasmática para análises moleculares.

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<li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rice+extracellular+vesicles+send+defense+proteins+into+fungus+rhizoctonia+solani+to+reduce+disease%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Rice extracellular vesicles send defense proteins into fungus rhizoctonia solani to reduce disease.</a> Dev Cell. 60 (8), 1168-1181.e6 (2025).</li>
<li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+mrnas+move+into+a+fungal+pathogen+via+extracellular+vesicles+to+reduce+infection%5BTitle%5D)+AND+%22Cell+Host+Microbe%22%5BJournal%5D)">Plant mrnas move into a fungal pathogen via extracellular vesicles to reduce infection.</a> Cell Host Microbe. 32 (1), 93-105.e6 (2024).</li>
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<li>Borniego, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diverse+plant+Rnas+coat+Arabidopsis+leaves+and+are+distinct+from+apoplastic+RNAs%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Diverse plant Rnas coat Arabidopsis leaves and are distinct from apoplastic RNAs.</a> Proc Natl Acad Sci U S A. 122 (1), e2409090121(2025).</li>
<li>Chen, A., He, B., Jin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+extracellular+vesicles+from+Arabidopsis%5BTitle%5D)+AND+%22Curr+Protoc%22%5BJournal%5D)">Isolation of extracellular vesicles from Arabidopsis.</a> Curr Protoc. 2 (1), e352(2022).</li>
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</ol>

<table><tbody><tr><td>0,45 & micro; Filtros de nylon m</td><td>VWR</td><td>76479-032</td><td></td></tr><tr><td>1 ml de seringa desnecessária</td><td>BD em Biociências</td><td>309659</td><td></td></tr><tr><td>Filtro ultra centrífugo de 100 kDa</td><td>Amicon</td><td>UFC 910008</td><td></td></tr><tr><td>100 ml seringa desnecessária</td><td>BD em Biociências</td><td>551721050</td><td></td></tr><tr><td>Tubos cônicos de 15 ml</td><td>VWR</td><td>89039-664</td><td></td></tr><tr><td>Tubos cônicos de 50 ml</td><td>VWR</td><td>89039-656</td><td></td></tr><tr><td>Arabidopsis thaliana (Col-0)</td><td>Linhas mantidas em laboratório</td><td>Não aplicável</td><td></td></tr><tr><td>Grades de 300 malhas carbono-formvar</td><td>TED PELLA</td><td>01753-F</td><td></td></tr><tr><td>Fita transparente</td><td>Fita Mágica Escocês</td><td>S-9783</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-12</td><td></td></tr><tr><td>Buffer PBS</td><td>Feito internamente</td><td>Não aplicável</td><td></td></tr><tr><td>qEV coluna original Gen2 35 nm</td><td>IZON Science</td><td>ICO-35</td><td></td></tr><tr><td>Centrífuga refrigerada</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Centrífuga refrigerada com rotor de ângulo fixo</td><td>Eppendorf</td><td>F-34-6-38</td><td></td></tr><tr><td>Rotor de balde oscilante refrigerado para centrífuga</td><td>Eppendorf</td><td>A-4-62</td><td></td></tr><tr><td>Sacarose</td><td>VWR</td><td>470302-810</td><td></td></tr><tr><td>Ultracentrífuga</td><td>Beckman Coulter</td><td>XPN-100</td><td></td></tr><tr><td>Tubos ultracentrífugas</td><td>Beckman Coulter</td><td>337986</td><td></td></tr><tr><td>Coloração UranyLess</td><td>Ciências da Microscopia Eletrônica</td><td>250218-01</td><td></td></tr></tbody></table>

isolamento e purificação de vesículas extracelulares vegetais de folhas de arabidopsis usando um método otimizado de coleta por lavagem apoplástica

Isolation and Purification of Plant Extracellular Vesicles from Arabidopsis Leaves Using an Optimized Apoplastic Wash Collection Method

Isolamento e purificação de vesículas extracelulares vegetais de folhas de Arabidopsis usando um método otimizado de coleta por lavagem apoplástica

Extracellular vesicles (EVs) are membrane-bound nanoparticles secreted by diverse organisms and cell types and are increasingly recognized as important ...

isolation-purification-plant-extracellular-vesicles-from-arabidopsis

Research

JoVE Journal

Biology

Biologia

Este vídeo mostra um procedimento para isolar protoplastos intactos a partir de tecidos de 14 dias de idade, mudas de Arabidopsis. Dado que o protoplastos isolados permanecem intactos por pelo menos 96h e são isoladas de mudas, em vez de um mês de idade plantas maduras, esse procedimento agiliza ensaios exigindo protoplastos intactos.

Isolamento de Protoplastos de tecidos de 14 dias de idade, mudas de Arabidopsis thaliana</em

Fonte: Rufián, J. S., et al, Análise de Célula Única da Expressão dos Genes de Pseudomonas syringae no Tecido Vegetal. J. Vis. Exp. (2022)Este vídeo demonstra um método de pressão baseado em seringa para extrair bactérias apoplásicas, que proliferam e formam microcolônias dentro do apoplasto, o espaço intercelular entre as células vegetais. O método fornece amostras bacterianas livres de detritos adequadas para análise de célula única a jusante.

Extração de Bactérias Apoplásticas Usando um Método de Pressão Baseado em Seringa

O conhecimento da composição da seiva floema, bem como o seu mecanismo de carregamento e de longa distância de transporte é essencial para a compreensão de sinalização de longa distância no desenvolvimento das plantas e stress / agente patogénico e da resposta assimilar transporte. Este manuscrito descreve a recolha de exsudados do floema, utilizando o método de EDTA-facilitada.

Coleta e análise de<em&gt; Arabidopsis</em&gt; Floema exsudados Usando o método EDTA facilitada

Este protocolo detalha a extração otimizada do fluido de lavagem do apoplasto das folhas das plantas, usando plantas de feijão francês (Phaseolus vulgaris) como exemplo modelo.

A técnica de infiltração-centrifugação para Extração de apoplásticas Fluid das folhas da planta Usando Phaseolus vulgaris Como exemplo

Aqui, apresentamos um protocolo de captura interativo aplicado aos protoplastos de mesofila da folha de Arabidopsis thaliana . Este método baseia-se criticamente na reticulação UV in vivo e permite o isolamento e identificação de proteínas de ligação a mRNA de plantas a partir de um ambiente fisiológico.

Capacidade de interactomo do mRNA a partir de protoplastos de plantas

Aqui, descrevemos um método para purificar cloroplastos intactos de Arabidopsis folhas e seus três principais compartimentos sub (envelope, estroma e contém), usando uma combinação de centrifugações diferenciais, contínuo de Percoll, e gradientes de sacarose descontínuo. O método é valioso para subplastidial e Localização subcellular das proteínas por immunoblotting e proteômica.

Preparação de cloroplasto sub compartimentos de Arabidopsis para a análise da localização da proteína pelo Immunoblotting ou Proteomics

Este protocolo isola vesículas extracelulares (EVs) longe de virions com alta eficiência e rendimento através da incorporação de precipitação EV, ultracentlee gradiente de densidade, e captura de partículas para permitir um fluxo de trabalho simplificado e uma redução de partida requisitos de volume, resultando em preparações reprodutíveis para uso em todas as pesquisas EV.

Purificação de preparações de vesículas extracelulares de alto rendimento longe do vírus

Um protocolo rápido e eficiente é apresentado para o isolamento de gotículas lipídicas de plastoglobule associadas a vários organismos fotossintéticos. A preparação bem-sucedida de plastoglóbulos isolados é um primeiro passo crucial que precede investigações moleculares detalhadas, como análises proteômicas e lipidômicas.

Isolamento de gotículas lipídicas de platoglobulos do tecido foliar da planta e cianobactérias

A purificação de proteínas recombinantes de sistemas vegetais é geralmente desafiada por proteínas vegetais. Este trabalho fornece um método para extrair e purificar efetivamente uma proteína recombinante secretada do apoplasto de Nicotiana benthamiana.

Método baseado em extração de apoplastos para melhorar a pureza da proteína recombinante produzida pela planta  

Descrevemos um método simples, mas altamente reprodutível, para isolar o RNA extracelular da superfície da folha e do apoplasto de plantas de Arabidopsis.

Um guia passo a passo para o isolamento e análise da superfície foliar e RNA apoplástico usando rosetas de Arabidopsis

Aprenda a isolar vesículas extracelulares de folhas de Arabidopsis thaliana usando infiltração suave e gradiente de sacarose. Este protocolo minimiza danos celulares e contaminação citoplasmática para análises moleculares.

Isolamento e purificação de vesículas extracelulares vegetais de folhas de Arabidopsis usando um método otimizado de coleta por lavagem apoplástica (Vídeo) | JoVE Journal

Isolamento e purificação de vesículas extracelulares vegetais de folhas de Arabidopsis usando um método otimizado de coleta por lavagem apoplástica

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