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<!DOCTYPE html>
<html lang="prt-br"> <!-- português -->
<head>
<meta charset="utf-8"> <!-- caracteres especiais -->
<meta http-equiv="X-UA-Compatible" content="IE=edge,chrome=1"> <!-- Permite usar o plug-in do Google Chrome que tem um mecanismo rápido de JavaScript ao abrir páginas no navegador -->
<title>FluxPRT</title> <!-- nome do Guia Interativo FluxPRT -->
<meta name="description" content="">
<meta name="viewport" content="width=device-width, initial-scale=1.0"> <!-- permite controlar a janela de visualização, reduzindo a página web inteira para caber na tela de celulares e ipads-->
<link rel="apple-touch-icon" href="apple-touch-icon.png"> <!-- Adapta os icones para vários dispositivos da apple-->
<!-- arquivos CSS -->
<link rel="stylesheet" href="css/bootstrap.min.css">
<link rel="stylesheet" href="css/bootstrap-theme.min.css">
<link rel="stylesheet" href="css/fontAwesome.css">
<link rel="stylesheet" href="css/light-box.css">
<link rel="stylesheet" href="css/owl-carousel.css">
<link rel="stylesheet" href="css/templatemo-style.css">
<link href="https://fonts.googleapis.com/css?family=Open+Sans:300,400,600,700,800" rel="stylesheet"> <!-- chamar a familia da fonte OpenSans -->
<script src="js/vendor/modernizr-2.8.3-respond-1.4.2.min.js"></script> <!-- biblioteca de js que detecta os recursos disponíveis no navegador e informa quando o navegador não é compatível -->
</head>
<body>
<header class="nav-down responsive-nav hidden-lg hidden-md">
<button type="button" id="nav-toggle" class="navbar-toggle" data-toggle="collapse" data-target="#main-nav">
<span class="sr-only">Toggle navigation</span> <!-- têcnica usada para acessibilidade-->
<span class="icon-bar"></span>
<span class="icon-bar"></span>
<span class="icon-bar"></span>
</button>
<!-- cabecalho da barra de navegação /navbar-header/ -->
<div id="main-nav" class="collapse navbar-collapse"><nav><ul class="nav navbar-nav">
<li><a href="#top">Extração de Proteínas</a></li>
<li><a href="#video">Fluxo de trabalho</a></li>
<li><a href="#blog">Protocolo de Extração</a></li>
<li><a href="#contact">e-book</a></li>
</ul></nav>
</div>
</header>
<div class="sidebar-navigation hidde-sm hidden-xs">
<div class="logo">
<a href="index.html"><em>Flux</em>PRT</a>
</div>
<nav>
<ul>
<li>
<a href="#video">
<em>Extração de Proteínas</em>
</a>
</li>
<li>
<a href="#video">
<span class="rect"></span>
<span class="circle"></span>
Fluxo de trabalho
</a>
</li>
</li>
<li>
<a href="#blog">
<span class="rect"></span>
<span class="circle"></span>
Protocolo de Extração
</a>
</li>
<li>
<a href="#contact">
<span class="rect"></span>
<span class="circle"></span>
e-book
</a>
</li>
</ul>
</nav>
<ul class="social-icons">
<!-- página inicial da página web -->
<li><a href="index.html"><i class="fa fa-home"></i></a></li>
<!-- github do guia FluxPRT -->
<li><a href="http://cabernet.luar.dcc.ufmg.br:7070/groups/luar" target="_blank"><i class="fa fa-github"></i></a></li>
<!-- webpage do LUAR -->
<li><a href="http://luar.dcc.ufmg.br/" target="_blank"><i class="fa fa-share-alt"></i></a></li>
<!-- email da desenvolvedora -->
<li><a href="mailto:elizabeth.espinoza@ufv.br" target="_blank"><i class="fa fa-envelope"></i></a></li>
</ul>
</div>
<div class="page-content">
<section id="video" class="content-section">
<div class="row"><div class="col-md-12"><div class="section-heading">
<h1>Você está na atividade <em>Extração de Proteínas</em> do FluxPRT</h1>
<p>O Guia Interativo do FluxPRT segue o Fluxo de Trabalho em Proteômica.</p>
</div>
<div class="text-content">
<p>O Guia Interativo do FluxPRT segue o Fluxo de Trabalho em Proteômica apresentado a seguir, clique nas atividades enumeradas mais detalhes e dicas. Aproveite instruções e dicas para tornar sua experiência com o FluxPRT ainda mais produtiva, nesta seção sao apresentadas informacões a respeito de Extração de Proteínas.<br><br></p>
</div>
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</section>
<section id="blog" class="content-section">
<div class="section-heading">
<h1><em>Protocolo</em><br>de Extração</h1><p>Para esta etapa essencial para a purificação de proteínas existem diferentes estratégias.<br>Aprenda mais sobre elas aqui:</p>
</div>
<div class="section-content"><div class="tabs-content"><div class="wrapper">
<ul class="tabs clearfix" data-tabgroup="first-tab-group">
<li><a href="#tab1" class="active">Vegetal</a></li>
<li><a href="#tab2">Animal</a></li>
<li><a href="#tab3">Fungi</a></li>
</ul>
<section id="first-tab-group" class="tabgroup">
<!-- vegetal -->
<div id="tab1"><ul><li><div class="item">
<div class="text-content">
<h4>Extração de Tecido Vegetal</h4>
<span>Fatores a considerar</span>
<p>As proteínas vegetais exigem condições específicas para sua extração e purificação pela sua ampla gama de proteínas com suas diferentes propriedades. Portanto, não é possível recomendar um único protocolo para extração de todas as proteínas vegetais (Doonan and Cutler, 2004). No entanto, os tecidos vegetais apresentam problemas específicos que podem ser levados em conta para o desenho do protocolo, veja aqui os principais:<br><ol>
<li><em>A presença da parede celular:</em> Pode se liberar as proteínas usando areia lavada com ácido junto com o tampão de extração e moagem em um pilão e argamassa ou adição de nitrogênio líquido para congelar rapidamente o material antes de misturar (Wang, Tai, and Chen, 2008;Wu, Gong, and Wang, 2014)</li>
<li><em>A presença de contaminantes específicos (compostos fenólicos, proteinases)</em>: Para evitar ou parcialmente controlar isto, pode se usar um tecido específico ou uma espécie vegetal específica. Mas não é possível controlar em alguns casos, nestes se recomenda encontrar maneiras de remover ou inativar os contaminantes ativos (Plaxton, 2019).</li>
<li><em>Baixo nível de proteínas (enzimas vegetais):</em> As proteínas vegetais podem estar presentes em níveis baixos em misturas altamente complexas. Uma exceção são os órgãos de armazenamento que foram tratados no capítulo anterior.</li>
</ol></p>
<p>Mas não tudo é coisa ruim, os e estudos comparativos da extração e solubilidade de proteínas vegetais feitos por Osborne entre 1880 e 1930, utilizando diversas fontes vegetais (Osborne and Harris, 1903), definiu quatro grupos extraídos sequencialmente em água (Osborne and Campbell, 1896:<br><ol>
<li><em>Albuminas</em>: soluções salinas diluídas.</li><br>
<li><em>Globulinas</em>: misturas álcool-água.</li><br>
<li><em>Prolaminas</em>: e diluir ácido ou álcali.</li><br>
<li><em>Glutelinas</em>: diluídas em ácido.</li>
</ol></p>
<p>Estes <em>grupos Osborne </em>ainda formam a base para estudos de proteínas de armazenamento de sementes e os termos albumina e globulina passaram a ser aceitos na comunidade científica.</p>
</div></div></li></ul></div>
<!-- //vegetal -->
<!-- animal -->
<div id="tab2"><ul><li><div class="item">
<div class="text-content">
<h4>Extração de Tecido Animal</h4>
<span>Conheça o seu experimento</span>
<p>Um dos primeiros passos a tomar em conta para isolar proteínas e preparar o extrato solúvel delas é conseguir romper os tecidos animais de tal maneira que a organela ou macromolécula de interesse possa ser purificada em um alto rendimento, livre de contaminantes e em uma forma ativa (Doonan and Cutler, 2004). A técnica empregada para isto é a de homogeneização, a qual deve de:<br><ol>
<li>Tensionar as células o suficiente para causar a ruptura da membrana plasmática superficial, liberando assim o citosol.</li>
<li>Não causar danos extensos às estruturas subcelulares, organelas e vesículas da membrana.</li>
</ol></p>
<p>A extração de proteínas de tecidos animais é mais simples em comparação com as paredes rígidas das células vegetais e de muitas bacterianas, pois elas apenas tem uma membrana plasmática suscetível às forças de cisalhamento. Dependendo se o tecido é mole (fígado e rim) ou não(esquelético e cardíaco), as forças deste requirirão ser mais suaves ou o contrário. Também deve ser considerada a distribuição subcelular das proteínas de interesse (Zabel and Klose, 2009). Veja aqui alguns exemplos:<br><ol>
<li><em>Organela específica (núcleo, mitocôndria, lisossomo ou retículo endoplasmático)</em>: Se recomenda isolar a organela fazendo um fracionamento subcelular inicial e simplificar nas etapas de purificação subsequentes (centrifugação) para evitar contaminar a amostra e a perda de proteínas. Para liberar as proteínas da organela, pode se tratar com detergente, por choque osmótico ou ultra-som.</li>
<li>N<em>a matriz extracelular (colágeno e elastina)</em>:As proteínas só podem ser solubilizadas após hidrólise química ou clivagem proteolítica (Schmidt, Dornelles, Mello, Kubota, Mazutti, Kempka, and Demiate, 2016).</li>
<li><em>Proteínas ligadas a membrana</em>: Podem ser as proteínas integradas no bocal fosfolipídeo hidrofóbico, ou associadas à membrana lipídica. As primeiras podem ser extraídas pela ruptura do bocal lipídico com um detergente ou com um solvente. Entanto, as segundas precisam ser liberadas de suas âncoras de membrana com uma protease adequada.</li>
</ol></p>
</div></div></li></ul></div>
<!-- //animal -->
<!-- fungi -->
<div id="tab3"><ul><li><div class="item">
<div class="text-content">
<h4>Extração de Proteínas Fúngicas</h4>
<span>Sobre a pureza da amostra</span>
<p>A extração de proteínas pode ser feita de quase qualquer tipo de material fúngico. Para extrair proteínas de leveduras e fungos filamentosos (Bridge, Kokubun, and Simmonds, 2004) é importante considerar:<br><ol>
<li>Verificar a localização das proteínas antes da extração, pois as proteínas extracelulares se perderão durante as extrações intracelulares.</li>
<li>A recuperação de proteínas grandes (150 kDa) após a ruptura física das células pode ser melhorada pela inclusão de inibidores de protease ou pelo uso de tampões específicos.</li>
</ol></p>
</div></div></li></ul></div>
<!-- //fungi -->
</section></div></div>
</div>
<br><br><strong>Referências</strong><br><br>
<p>Shawn Doonan and Paul Cutler. <a href="http://link.springer.com/10.1385/1-59259-655-X:1" target="_blank">General Strategies</a>. <em>In Protein Purification Protocols</em>, chapter 1, pages 1–14. Humana Press, New Jersey, 2004. doi: 10.1385/1-59259-655-X:1</p>
<p>Wei Wang, Fuju Tai, and Shaoning Chen. <a href="https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/abs/10.1002/jssc.200800087" target="_blank">Optimizing protein extraction from plant tissues for enhanced proteomics analysis</a>. <em>Journal of Separation Science</em>, 31(11):2032–2039, 2008. doi: 10.1002/jssc.200800087</p>
<p>Xiaolin Wu, Fangping Gong, and Wei Wang. <a href="https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/abs/10.1002/pmic.201300239" target="_blank">Protein extraction from plant tissues for 2DE and its application in proteomic analysis</a>. Proteomics, 14(6):645–658, 2014. doi: 10.1002/pmic.201300239</p>
<p>William C Plaxton. <a href="https://doi.org/10.1093/pcp/pcz028" target="_blank">Avoiding Proteolysis during the Extraction and Purification of Active Plant Enzymes</a>. <em>Plant and Cell Physiology</em>, 60(4):715–724, 02 2019. ISSN 0032-0781. doi: 10.1093/pcp/pcz028.</p>
<p>Thomas B Osborne and Isaac F Harris. <a href="https://doi.org/10.1021/ja02010a008" target="_blank">The precipitation limits with ammonium sulphate of some vegetable proteins</a>. <em>Journal of the American Chemical Society</em>, 25(8):837–842, 1903. ISSN 0002-7863. doi: 10.1021/ja02010a008</p>
<p>Thomas B Osborne and George F Campbell. <a href="https://doi.org/10.1021/ja02093a004" target="_blank">Conglutin and vitellin</a>. <em>Journal of the American Chemical Society</em>, 18(7):609–623, 1896. ISSN 0002-7863. doi: 10.1021/ja02093a004</p>
<p>Claus Zabel and Joachim Klose. <a href="https://doi.org/10.1007/978-1-59745-198-7_12" target="_blank">Mouse and human tissue sample preparation for 2-D electrophoresis</a>. <em>In The Protein Protocols Handbook</em>, pages 85–107. Humana Press, Totowa, NJ, 2009. ISBN 978-1-59745-198-7. doi: 10.1007/978-1-59745-198-7_12</p>
<p>MM Schmidt, RCP Dornelles, RO Mello, EH Kubota, MA Mazutti, AP Kempka, and IM Demiate. <a href="http://ifrj.upm.edu.my/23%20(03)%202016/(1).pdf" target="_blank"><em>Collagen extraction process</em></a>. International Food Research Journal, 23(3), 2016.</p>
<p>Paul D Bridge, Tetsuo Kokubun, and Monique SJ Simmonds. <a href="https://doi.org/10.1385/1-59259-655-X:37" target="_blank">Protein extraction from fungi</a>.<em> In Protein Purification Protocols</em>, pages 37–46. Humana Press, 2004. ISBN 978-1-59259-655-3. doi:10.1385/1-59259-655-X:37.</p>
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