{"id":1990,"date":"2025-01-03T16:45:00","date_gmt":"2025-01-03T16:45:00","guid":{"rendered":"https:\/\/www.gvs.com.br\/life-sciences\/?p=1990"},"modified":"2025-10-07T22:21:15","modified_gmt":"2025-10-07T22:21:15","slug":"western-blotting","status":"publish","type":"post","link":"https:\/\/www.gvs.com.br\/life-sciences\/2025\/01\/03\/western-blotting\/","title":{"rendered":"Western Blotting: saiba tudo sobre as etapas desse teste"},"content":{"rendered":"\n<div class=\"wp-block-uagb-container uagb-block-8556d22c alignfull uagb-is-root-container\"><div class=\"uagb-container-inner-blocks-wrap\">\n<div class=\"wp-block-uagb-advanced-heading uagb-block-62d72bed\"><h1 class=\"uagb-heading-text\">Western Blotting: saiba tudo sobre as etapas <br>desse teste<\/h1><\/div>\n\n\n\n<div class=\"wp-block-uagb-advanced-heading uagb-block-cca09862\"><h2 class=\"uagb-heading-text\">Descubra o passo a passo desse m\u00e9todo e qual a sua import\u00e2ncia dele para a obten\u00e7\u00e3o de resultados.<\/h2><\/div>\n<\/div><\/div>\n\n\n\n<p><\/p>\n\n\n\n<p>Em nosso blog, j\u00e1 falamos sobre a t\u00e9cnica de pesquisa Western Blotting; especificamente sobre suas aplica\u00e7\u00f5es e objetivo.<\/p>\n\n\n\n<p>Para aprofundar ainda mais o tema, hoje explicaremos quais as principais etapas do processo, como s\u00e3o realizadas e qual a fun\u00e7\u00e3o de cada passo.<\/p>\n\n\n\n<p>Se tem d\u00favidas sobre o assunto, continue a leitura e descubra tudo sobre esse meio de an\u00e1lise biol\u00f3gica!<\/p>\n\n\n\n<h2 class=\"wp-block-heading has-ast-global-color-0-color has-text-color has-link-color wp-elements-3e511dbc18e400c4e2e0dd2fcfab8727\">O que \u00e9 Western Blotting?<\/h2>\n\n\n\n<p>A principal fun\u00e7\u00e3o da transfer\u00eancia Western, com tamb\u00e9m \u00e9 chamada, \u00e9 detectar e quantificar prote\u00ednas presentes no componente biol\u00f3gico de estudo.<\/p>\n\n\n\n<p>Desse modo, facilita-se a observa\u00e7\u00e3o de resultados de uma pesquisa como, por exemplo, o aumento de prote\u00ednas de uma amostra espec\u00edfica.<\/p>\n\n\n\n<p>Como j\u00e1 citado, esse procedimento, comum em ambientes de pesquisa, possui etapas a serem seguidas, para que seja realizado com o m\u00e1ximo de efici\u00eancia.<\/p>\n\n\n\n<p>Entre essas etapas, podemos citar:<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li>Extra\u00e7\u00e3o e quantifica\u00e7\u00e3o das prote\u00ednas;<\/li>\n\n\n\n<li>Fracionamento das prote\u00ednas da amostra em um gel de poliacrilamida; &nbsp;<\/li>\n\n\n\n<li>Transfer\u00eancias dessas prote\u00ednas para uma membrana; &nbsp;<\/li>\n\n\n\n<li>Incuba\u00e7\u00e3o da membrana com um anticorpo para detectar a prote\u00edna;<\/li>\n\n\n\n<li>Detec\u00e7\u00e3o da prote\u00edna espec\u00edfica.<\/li>\n<\/ul>\n\n\n\n<p>Devido a import\u00e2ncia de realizar o procedimento seguindo todos os passos, para garantir bons resultados, \u00e9 necess\u00e1rio saber qual a finalidade de cada etapa.<\/p>\n\n\n\n<p>Ent\u00e3o, continue a leitura!<\/p>\n\n\n\n<h2 class=\"wp-block-heading has-ast-global-color-0-color has-text-color has-link-color wp-elements-f45520b6ae8defa793e34c219b502142\">Extra\u00e7\u00e3o e quantifica\u00e7\u00e3o das prote\u00ednas<\/h2>\n\n\n\n<p>O primeiro passo quando falamos do experimento Western Blotting, \u00e9 a extra\u00e7\u00e3o e quantifica\u00e7\u00e3o das prote\u00ednas.<\/p>\n\n\n\n<p>Isso porque, \u00e9 o passo inicial para analisar a presen\u00e7a ou n\u00e3o de prote\u00ednas de uma amostra e sua quantidade.<\/p>\n\n\n\n<p>O ensaio Bradford, \u00e9 a maneira mais utilizada para obten\u00e7\u00e3o dos resultados necess\u00e1rios; isso porque, apresenta diversas vantagens em rela\u00e7\u00e3o a outros dispon\u00edveis.<\/p>\n\n\n\n<p>O procedimento \u00e9 comum, por sua velocidade para a chegada aos resultados. Al\u00e9m disso, n\u00e3o exige aquecimento ou alta sensibilidade, sendo capaz de agir sobre baixas quantidades de prote\u00edna na amostra.<\/p>\n\n\n\n<p>O m\u00e9todo se baseia na mudan\u00e7a de cor do composto Coomassie Blue G-250, que apresenta cor avermelhada para solu\u00e7\u00f5es \u00e1cidas, e muda para a cor azul ao entrar em contato com as prote\u00ednas da amostra.<\/p>\n\n\n\n<p>Ap\u00f3s constatada a exist\u00eancia de prote\u00ednas no objeto de an\u00e1lise, \u00e9 necess\u00e1rio quantificar a sua presen\u00e7a.<\/p>\n\n\n\n<p>Por isso, \u00e9 utilizada uma curva de padroniza\u00e7\u00e3o, usando concentra\u00e7\u00f5es j\u00e1 conhecidas de outra prote\u00edna.<\/p>\n\n\n\n<p>Dessa forma, obtemos uma equa\u00e7\u00e3o linear em que, trocando os valores m\u00e9dios de absorb\u00e2ncia das amostras, chegamos aos valores de concentra\u00e7\u00e3o da prote\u00edna.<\/p>\n\n\n\n<h2 class=\"wp-block-heading has-ast-global-color-0-color has-text-color has-link-color wp-elements-c813a7ff131c8150ee491cb15ad02e9f\">Fracionamento das prote\u00ednas<\/h2>\n\n\n\n<p>Agora que j\u00e1 contatamos, como \u00e9 realizada a extra\u00e7\u00e3o e quantifica\u00e7\u00e3o de uma prote\u00edna em amostra, \u00e9 preciso entender como \u00e9 realizado o fracionamento dessas subst\u00e2ncias e qual a raz\u00e3o do procedimento.<\/p>\n\n\n\n<p>O m\u00e9todo de fracionamento mais comum, \u00e9 a eletroforese em gel de SDS-poliacrilamida.<\/p>\n\n\n\n<p>Nesse processo, as mol\u00e9culas que possuem cargas positivas e negativas, como as prote\u00ednas, s\u00e3o separadas de acordo com a suas caracter\u00edsticas f\u00edsicas e peso molecular, quando passam por uma corrente el\u00e9trica.<\/p>\n\n\n\n<p>Dessa forma, o seu uso se torna comum. Pois, \u00e9 respons\u00e1vel pela separa\u00e7\u00e3o, at\u00e9 mesmo, de prote\u00ednas insol\u00faveis em \u00e1gua, caso comum em muitas prote\u00ednas de membrana, por exemplo.<\/p>\n\n\n\n<p>Al\u00e9m disso, com a separa\u00e7\u00e3o feita atrav\u00e9s do tamanho das mol\u00e9culas, \u00e9 poss\u00edvel determinar o peso molecular e a composi\u00e7\u00e3o da prote\u00edna.<\/p>\n\n\n\n<p>Outro fator importante, \u00e9 a possibilidade de visualizar a prote\u00edna por meio do uso de corantes ou tratamento com nitrato de prata.<\/p>\n\n\n\n<p>Por isso, a colora\u00e7\u00e3o do componente \u00e9 alterada, o que facilita a percep\u00e7\u00e3o da sua presen\u00e7a na amostra analisada.<\/p>\n\n\n\n<p>Nos casos em que o nitrato de prata \u00e9 usado, \u00e9 poss\u00edvel identificar a presen\u00e7a de prote\u00ednas em baixa concentra\u00e7\u00e3o, j\u00e1 que \u00e9 um m\u00e9todo mais sens\u00edvel.<\/p>\n\n\n\n<p>Os corantes espec\u00edficos tamb\u00e9m s\u00e3o bem comuns, principalmente, por sua praticidade, como exemplo, podemos citar o\u00a0<a href=\"https:\/\/www.gvs.com.br\/life-sciences\/biologia-molecular-lightwave\/\" target=\"_blank\" rel=\"noreferrer noopener\">Lightwave<\/a>\u00a0da GVS, trata-se de um substrato a base de luminol n\u00e3o ist\u00f3pico.<\/p>\n\n\n\n<p>O componente \u00e9 projetado para quimioluminescentes detec\u00e7\u00e3o de prote\u00ednas imobilizadas e \u00e1cidos nucl\u00e9icos imobilizados conjugados com peroxidase de r\u00e1bano.<\/p>\n\n\n\n<figure class=\"wp-block-image aligncenter size-large\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"323\" src=\"https:\/\/www.gvs.com.br\/life-sciences\/wp-content\/uploads\/2025\/10\/meio-life-science-western-blotting-1024x323.jpg\" alt=\"\" class=\"wp-image-1996\" srcset=\"https:\/\/www.gvs.com.br\/life-sciences\/wp-content\/uploads\/2025\/10\/meio-life-science-western-blotting-1024x323.jpg 1024w, https:\/\/www.gvs.com.br\/life-sciences\/wp-content\/uploads\/2025\/10\/meio-life-science-western-blotting-300x95.jpg 300w, https:\/\/www.gvs.com.br\/life-sciences\/wp-content\/uploads\/2025\/10\/meio-life-science-western-blotting-768x242.jpg 768w, https:\/\/www.gvs.com.br\/life-sciences\/wp-content\/uploads\/2025\/10\/meio-life-science-western-blotting.jpg 1110w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><\/figure>\n\n\n\n<h2 class=\"wp-block-heading has-ast-global-color-0-color has-text-color has-link-color wp-elements-9272cb4952502a87a6907533274d4ea2\">Transfer\u00eancia para membrana<\/h2>\n\n\n\n<p>Ap\u00f3s a fragmenta\u00e7\u00e3o das prote\u00ednas presentes em uma solu\u00e7\u00e3o, \u00e9 necess\u00e1rio realizar a transfer\u00eancia do material obtido para uma&nbsp;<a href=\"https:\/\/www.gvs.com.br\/life-sciences\/biologia-molecular-membranas-de-transferencia-pvdf-pure\/\" target=\"_blank\" rel=\"noreferrer noopener\">membrana de transfer\u00eancia<\/a>.<\/p>\n\n\n\n<p>Para isso, existem diversos m\u00e9todos utilizados como: v\u00e1cuo, capilaridade e difus\u00e3o, mas, o processo mais comum nesse tipo de an\u00e1lise \u00e9 a eletroelui\u00e7\u00e3o ou&nbsp;<em>electroblotting<\/em>.<\/p>\n\n\n\n<p>Com o aux\u00edlio de almofadas porosas e papel de filtro, \u00e9 feita a aplica\u00e7\u00e3o de uma carga el\u00e9trica, que resulta na sa\u00edda da prote\u00edna do gel de poliacrilamida.<\/p>\n\n\n\n<p>Assim, as prote\u00ednas se ligam \u00e0 superf\u00edcie da membrana, mantendo o padr\u00e3o estabelecido no gel de poliacrilamida.<\/p>\n\n\n\n<p>A efici\u00eancia do procedimento \u00e9 dependente de alguns fatores como, por exemplo, a composi\u00e7\u00e3o do gel, presen\u00e7a de detergentes e \u00e1lcool no tamp\u00e3o, e a composi\u00e7\u00e3o das prote\u00ednas.<\/p>\n\n\n\n<p>Al\u00e9m disso, o tempo de transfer\u00eancia exerce forte influ\u00eancia nos resultados do processo, assim como, o contato da membrana com o gel, que deve ser completo.<\/p>\n\n\n\n<p>Ap\u00f3s a realiza\u00e7\u00e3o do procedimento de transfer\u00eancia, existem alguns processos a serem seguidos para confirmar a passagem das prote\u00ednas do gel para a membrana.<\/p>\n\n\n\n<p>Nesse passo, a utiliza\u00e7\u00e3o de corantes que reagem \u00e0 prote\u00edna \u00e9 muito utilizado para constatar a aus\u00eancia de prote\u00edna no gel de poliacrilamida.<\/p>\n\n\n\n<p>Al\u00e9m do mais, \u00e9 poss\u00edvel verificar a igualdade entre as amostras resultantes da transfer\u00eancia e, apenas em situa\u00e7\u00f5es satisfat\u00f3rias, \u00e9 dado seguimento ao processo de Western Blotting.<\/p>\n\n\n\n<h2 class=\"wp-block-heading has-ast-global-color-0-color has-text-color has-link-color wp-elements-c92bb3835f49467654e39e083743150f\">Incuba\u00e7\u00e3o da membrana<\/h2>\n\n\n\n<p>Depois de passar por extra\u00e7\u00e3o, quantifica\u00e7\u00e3o, fracionamento e transfer\u00eancia, a prote\u00edna j\u00e1 na membrana, passa por um processo de incuba\u00e7\u00e3o.<\/p>\n\n\n\n<p>Esse procedimento, se obt\u00e9m por meio da aplica\u00e7\u00e3o de uma prote\u00e7\u00e3o, para que os anticorpos n\u00e3o se liguem a toda superf\u00edcie da membrana.<\/p>\n\n\n\n<p>Assim, garantindo a presen\u00e7a deles apenas no ant\u00edgeno, e bloqueando o restante da membrana.<\/p>\n\n\n\n<p>Normalmente, a utiliza\u00e7\u00e3o de leite em p\u00f3 desnatado \u00e9 o mais indicado para esse bloqueio, exceto nos casos em que os componentes do leite podem prejudicar a liga\u00e7\u00e3o dos anticorpos aos ant\u00edgenos.<\/p>\n\n\n\n<p>Nesses casos, \u00e9 utilizado o PVP (poli-vinil-pilorridona), que \u00e9 um pol\u00edmero sint\u00e9tico que n\u00e3o prejudica essa rela\u00e7\u00e3o.<\/p>\n\n\n\n<p>Ap\u00f3s a incuba\u00e7\u00e3o com a solu\u00e7\u00e3o inicial, que deve durar cerca de uma hora e estar em temperatura ambiente e constante agita\u00e7\u00e3o, \u00e9 realizada a lavagem da membrana tr\u00eas vezes a cada 5 minutos.<\/p>\n\n\n\n<p>Depois disso, \u00e9 iniciado o processo de adi\u00e7\u00e3o de anticorpos a membrana, ou seja, o primeiro componente a se ligar ao ant\u00edgeno.<\/p>\n\n\n\n<p>Para isso, \u00e9 realizada a dilui\u00e7\u00e3o dos anticorpos \u00e0 solu\u00e7\u00e3o de bloqueio. Alguns protocolos, padronizam a fra\u00e7\u00e3o de 1\/100 para a realiza\u00e7\u00e3o dessa mistura.<\/p>\n\n\n\n<p>Assim, ap\u00f3s a aplica\u00e7\u00e3o da dilui\u00e7\u00e3o na membrana, \u00e9 feita uma nova incuba\u00e7\u00e3o, por uma hora e em constante agita\u00e7\u00e3o.<\/p>\n\n\n\n<p>O segundo anticorpo obtido, \u00e9 a mol\u00e9cula de ant\u00edgeno; esse anticorpo pode conservar a fun\u00e7\u00e3o dos dois componentes, como a atividade enzim\u00e1tica da enzima e antigenicidade do ant\u00edgeno.<\/p>\n\n\n\n<p>Al\u00e9m disso, ele \u00e9 classificado como anti-IgG, ou seja, ele pode localizar outros anticorpos com a mesma estrutura.<\/p>\n\n\n\n<p>Existem algumas modifica\u00e7\u00f5es nesse segundo anticorpo, que permitem a visualiza\u00e7\u00e3o da prote\u00edna de interesse, \u00e1 qual ele \u00e9 submetido.<\/p>\n\n\n\n<h2 class=\"wp-block-heading has-ast-global-color-0-color has-text-color has-link-color wp-elements-6c3a4eed62bbdb3abe1d60e9a2b6ba43\">Quais as aplica\u00e7\u00f5es do m\u00e9todo Western Blotting?<\/h2>\n\n\n\n<p>Como j\u00e1 mencionamos, alguns passos presentes no procedimento, permitem um resultado mais r\u00e1pido e com menos requisitos para serem alcan\u00e7ados.<\/p>\n\n\n\n<p>Dessa forma, sua utiliza\u00e7\u00e3o \u00e9 muito comum em diversos setores que necessitam de an\u00e1lise biol\u00f3gica como, por exemplo, testes que detectam a presen\u00e7a de HIV.<\/p>\n\n\n\n<p>Ali\u00e1s, o teste Western Blotting \u00e9 o meio confirmat\u00f3rio de HIV, j\u00e1 que detecta a presen\u00e7a de anticorpos ao v\u00edrus.<\/p>\n\n\n\n<p>Mas, n\u00e3o se resume apenas a isso, ele pode ser realizado para diagn\u00f3sticos de diversas doen\u00e7as e processos que necessitam de uma dimens\u00e3o para a presen\u00e7a de prote\u00ednas em uma solu\u00e7\u00e3o, por exemplo.<\/p>\n\n\n\n<p>Gostou do tema e tem mais d\u00favidas sobre processos parecidos?<\/p>\n\n\n\n<p>Acompanhe&nbsp;<a href=\"https:\/\/www.gvs.com.br\/life-sciences\/blog\/\" target=\"_blank\" rel=\"noreferrer noopener\">nosso blog<\/a>&nbsp;e leia mais mat\u00e9rias como esta!<\/p>\n\n\n\n<p><a href=\"https:\/\/kasvi.com.br\/western-blotting-analise-proteinas\/\">https:\/\/kasvi.com.br\/western-blotting-analise-proteinas\/<\/a><\/p>\n\n\n\n<p><a href=\"http:\/\/www.conhecer.org.br\/enciclop\/2012b\/ciencias%20agrarias\/western.pdf\">http:\/\/www.conhecer.org.br\/enciclop\/2012b\/ciencias%20agrarias\/western.pdf<\/a><\/p>\n\n\n\n<div class=\"wp-block-uagb-separator uagb-block-6ec145c0\"><div class=\"uagb-separator-spacing-wrapper\"><div class=\"wp-block-uagb-separator__inner\" style=\"--my-background-image:\"><\/div><\/div><\/div>\n\n\n\n<div class=\"wp-block-uagb-call-to-action uagb-block-da331e7c wp-block-button\"><div class=\"uagb-cta__wrap\"><h3 class=\"uagb-cta__title\">Fale com a gente!<\/h3><p class=\"uagb-cta__desc\">Entre em contato com o nosso time hoje mesmo, esclare\u00e7a as suas d\u00favidas e <strong>solicite a sua cota\u00e7\u00e3o!<\/strong><\/p><\/div><div class=\"uagb-cta__buttons\"><a href=\"https:\/\/wa.me\/5519997710045\" class=\"uagb-cta__button-link-wrapper wp-block-button__link\" target=\"_blank\" rel=\"noopener noreferrer\">WhastApp<\/a><\/div><\/div>\n\n\n\n<div class=\"wp-block-uagb-separator uagb-block-ee9b8592\"><div class=\"uagb-separator-spacing-wrapper\"><div class=\"wp-block-uagb-separator__inner\" style=\"--my-background-image:\"><\/div><\/div><\/div>\n\n\n\n<div class=\"wp-block-uagb-call-to-action uagb-block-5a340998 wp-block-button\"><div class=\"uagb-cta__wrap\"><h3 class=\"uagb-cta__title\">Fique por dentro das novidades!<\/h3><p class=\"uagb-cta__desc\">Acesse e conhe\u00e7a a Linha Completa de<strong>&nbsp;Life Sciences da GVS<\/strong>.<\/p><\/div><div class=\"uagb-cta__buttons\"><a href=\"https:\/\/www.gvs.com.br\/life-sciences\" class=\"uagb-cta__button-link-wrapper wp-block-button__link\" target=\"_blank\" rel=\"noopener noreferrer\">Acesse J\u00e1!<\/a><\/div><\/div>\n\n\n\n<p><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Western Blotting: saiba tudo sobre as etapas desse teste Descubra o passo a passo desse m\u00e9todo e qual a sua [&hellip;]<\/p>\n","protected":false},"author":2,"featured_media":1995,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_uag_custom_page_level_css":"","site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"disabled","ast-breadcrumbs-content":"","ast-featured-img":"disabled","footer-sml-layout":"","ast-disable-related-posts":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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