Name: ultra-high-speed western blot using immunoreaction enhancing technology
Uploaded: 2020-09-26T13:00:00.000+00:00
Duration: 5 min 59 s
Description: Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Este trabalho foi apoiado pela Divisão de Pesquisa Intramuros, Instituto Nacional de Coração, Pulmão e Sangue, Institutos Nacionais de Saúde. A S.H. foi apoiada pelo Programa de Estudos de Estudantes de Parceria Público-Privada do Japão no Exterior, e H.N. e K.Y. foram pela Valor e V Drug Overseas Training Scholarship.

1. SDS-PAGE e transferência para uma membrana PVDF
<ol><li>Execute o SDS-PAGE para separar proteínas com base no tamanho relativo. NOTA: Qualquer sistema de gel comercial ou caseiro está bem. Por favor, siga o protocolo do fabricante.</li>
	<li>Transfira proteínas separadas de um gel para uma membrana PVDF. NOTA: Os métodos comuns de transferência (transferência semi-seca ou úmida) são adequados. Por favor, siga o protocolo do fabricante.</li>
</ol>2. Bloqueio de membranas PVDF
<ol><li>Incubar as membranas em tampões de bloqueio apropriados por 1h com agitação à temperatura ambiente. NOTA: Dependendo do sistema de anticorpos e detecção primário, um buffer de bloqueio apropriado deve ser selecionado. Por exemplo, os bloqueadores mais típicos são BSA, leite seco sem gordura, caseína e polímeros sintéticos comerciais. PBS e/ou TBS com 0,1% Tween20 são os buffers mais utilizados. Esta etapa de bloqueio pode ser feita durante a noite a 4 °C.</li>
</ol>3. Incubação com anticorpo primário
<ol><li>Prepare 10% de imunoreaction melhorando a solução agent-1 (IRE-1) com água destilada. Vórtice bem.</li>
	<li>Prepare o anticorpo primário diluído com 10% de IRE-1. Misture-o suavemente e bem.</li>
	<li>Se usar uma membrana maior (4 cm x 8 cm – 8 cm x 8 cm), adicione 8 mL da solução de anticorpos em tubos de centrífuga cônica de 50 mL. Se utilizar uma membrana menor (2 cm x 8 cm – 4 cm x 8 cm), adicione 3 mL da solução de anticorpos em tubo de polipropileno de fundo redondo de 14 mL(Tabela de Materiais).</li>
	<li>Pegue a membrana PVDF com pinças e drene brevemente a solução de bloqueio. Insira a membrana PVDF neste tubo com os dedos enluvados e certifique-se de que toda a membrana adere à parede do tubo. Quando a membrana PVDF é inserida em um tubo, encare o "lado proteico" da membrana que estava originalmente em contato com o gel para dentro. Feche a tampa com força.</li>
	<li>Insira o tubo de 50 mL em uma garrafa de vidro para o forno de hibridização. NOTA: Quando forem utilizados tubos de 14 mL para esta incubação, insira um tubo de 14 mL no tubo de 50 mL, utilizando um par de suportes de anel plástico para manter o tubo interno no centro do tubo de 50 mL.</li>
	<li>Ligue o forno de hibridização.</li>
	<li>Incubar a membrana com rotação de 6 rpm por pelo menos 5 min. NOTA: Certifique-se de que a membrana adere à parede o tempo todo e que o tubo (interno) esteja horizontal para cobrir a membrana uniformemente com a solução de anticorpos. Calibrar a diluição do anticorpo e o tempo de incubação antes do experimento real.</li>
</ol>4. Lavagem de membrana
<ol><li>Pare a rotação.</li>
	<li>Remova cuidadosamente a membrana PVDF do tubo com pinças e coloque a membrana em um recipiente com 50 mL de PBS-T.</li>
	<li>Enxágüe a membrana brevemente com PBS-T.</li>
	<li>Enxágüe a membrana no recipiente com água destilada até que não apareçam bolhas. Isto é para remover a maioria do anticorpo.</li>
	<li>Transfira a membrana PVDF do recipiente para o rotador de salada que contém 250 mL de PBS-T.</li>
	<li>Coloque a cesta de coador no rotador. Certifique-se de que a tampa foi colocada com segurança.</li>
	<li>Ative o rotador e execute-o por cerca de 20-30 s.</li>
	<li>Descarte a solução e enxágue brevemente o interior do rotador com água destilada.</li>
	<li>Adicione 250 mL de PBS-T no spinner novamente.</li>
	<li>Repita as etapas 4.6 e 4.7.</li>
</ol>5. Incubação com o anticorpo secundário
<ol><li>Prepare 10% de imunoreaction melhorando a solução agent-2 (IRE-2) com água destilada. Vórtice bem.</li>
	<li>Prepare o anticorpo secundário diluído com 10% de IRE-2. Misture-o suavemente e bem.</li>
	<li>Selecione o volume da solução de anticorpos e do tubo conforme indicado na etapa 3.3.</li>
	<li>Pegue a membrana PVDF com pinças e drene a solução brevemente. Insira a membrana PVDF no tubo conforme indicado na etapa 3.4.</li>
	<li>Insira o tubo de 50 mL em uma garrafa de vidro para o forno de hibridização.</li>
	<li>Ligue o forno de hibridização.</li>
	<li>Incubar a membrana com rotação de 6 rpm por pelo menos 5 min. Certifique-se de que a membrana adere à parede o tempo todo e que o tubo (interno) esteja horizontal para cobrir uniformemente a membrana com a solução de anticorpos. NOTA: Calibrar o tempo de incubação antes do experimento real. Quando o anticorpo secundário for fluorescentemente conjugado, realize a incubação no escuro.</li>
</ol>6. Lavagem de membrana
<ol><li>Siga os passos 4.1 às 4:10.</li>
</ol>7. Aquisição de imagens
<ol><li>Detecção de chemiluminescente<ol>
<li>Coloque um pedaço de filme flexível semi-transplante (10 cm x 15 cm) em uma superfície plana.</li>
		<li>Misture 2 componentes do substrato chemiluminescente em um tubo de 5 mL.</li>
		<li>Transfira imediatamente 1,5 mL do substrato misto para a filmagem flexível semi-transplante e coloque a membrana PVDF sobre ele. Em seguida, transfira o resto da solução do substrato para a membrana.</li>
		<li>Incubar a membrana PVDF com o substrato misto em uma película flexível semi-transparente por 1 min.</li>
		<li>Pegue a membrana PVDF com pinças e escorra brevemente a solução do substrato.</li>
		<li>Sanduíche a membrana entre um par de filmes de transparência.</li>
		<li>Adquira a imagem sob o modo chemiluminescent.</li>
	</ol>
</li>
	<li>Detecção fluorescente<ol>
<li>Pegue a membrana PVDF com pinças e drene a solução brevemente.</li>
		<li>Sanduíche a membrana entre um par de filmes de transparência.</li>
		<li>Adquira a imagem sob o modo fluorescente. NOTA: As etapas 7.1 e 7.2 devem ser realizadas nas proximidades da máquina de imagem para garantir a máxima sensibilidade.</li>
		<li>Quando o sinal de fundo estiver alto, realize um passo de lavagem em um recipiente com 80-100 mL de PBS-T: 10 min enxágue 3 vezes para o anticorpo primário e 5 min enxágue 6 vezes para o anticorpo secundário. NOTA: Tanto os anticorpos primários quanto secundários diluídos com soluções IRE de 10% podem ser usados até 8-10 vezes sem perda de sensibilidade6. A solução deve ser mantida em 4 °C por até 1 mês.</li>
	</ol>
</li>
</ol>

<ol>
	<li>MacPhee, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+for+improving+Western+blot+analysis.">Methodological considerations for improving Western blot analysis.</a> Journal of Pharmacol Toxicology Methods. 61 (2), 171-177 (2010).</li><li>Janes, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+critical+factors+for+quantitative+immunoblotting.">An analysis of critical factors for quantitative immunoblotting.</a> Science Signaling. 8 (371), (2015).</li><li>Schuck, P., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mass+transport+limitation+and+surface+heterogeneity+in+the+biophysical+characterization+of+macromolecular+binding+processes+by+SPR+biosensing.">The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing.</a> Methods in Molecular Biology. 627, 15-54 (2010).</li><li>Li, J., Zrazhevskiy, P., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eliminating+Size-Associated+Diffusion+Constraints+for+Rapid+On-Surface+Bioassays+with+Nanoparticle+Probes.">Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes.</a> Small. 12 (8), 1035-1043 (2016).</li><li>Higashi, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Old+but+not+Obsolete:+An+Enhanced+High+Speed+Immunoblot.">Old but not Obsolete: An Enhanced High Speed Immunoblot.</a> Journal of Biochemistry. 168 (1), 15-22 (2020).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+Procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications.</a> Proceedings of the National Academy of Sciences of the United States of America. 76, 4350-4354 (1979).</li><li>Burnette, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Western+blotting":+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112 (2), 195-203 (1981).</li><li>Mishra, M., Tiwari, S., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification+and+analysis:+next+generation+Western+blotting+techniques.">Protein purification and analysis: next generation Western blotting techniques.</a> Expert Review of Proteomics. 14 (11), 1037-1053 (2017).</li><li>Ciaccio, M. F., Wagner, J. P., Chuu, C. P., Lauffenburger, D. A., Jones, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+analysis+of+EGF+receptor+signaling+dynamics+with+microwestern+arrays.">Systems analysis of EGF receptor signaling dynamics with microwestern arrays.</a> Nature Methods. 7 (2), 148-155 (2010).</li><li>Treindl, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bead-based+western+for+high-throughput+cellular+signal+transduction+analyses.">A bead-based western for high-throughput cellular signal transduction analyses.</a> Nature Communications. 7, 12852 (2016).</li><li>Sajjad, S., Do, M. T., Shin, H. S., Yoon, T. S., Kang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+western+blot+assay+by+rotational+cyclic+draining+and+replenishing+procedure.">Rapid and efficient western blot assay by rotational cyclic draining and replenishing procedure.</a> Electrophoresis. 39 (23), 2974-2978 (2018).</li><li>Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+review+of+other+notable+protein+detection+methods+on+blots.">A brief review of other notable protein detection methods on blots.</a> Methods in Molecular Biology. 536, 557-571 (2009).</li><li>Nagase, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+and+Sensitive+Detection+of+Glycosaminoglycan+Chains+with+Immunoblots.">Reliable and Sensitive Detection of Glycosaminoglycan Chains with Immunoblots.</a> Glycobiology. , (2020).</li></ol>

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A mancha ocidental é uma técnica analítica amplamente utilizada para detectar proteínas específicas que foram desenvolvidas ~40 anos atrás7,8. Desde então, a técnica continuou a evoluir, com inovações subsequentes melhorando a sensibilidade, velocidade e quantitação da técnica2,9,10,11,12<...

Uma mancha ocidental (também conhecida como imunoblot) é uma técnica poderosa e fundamental em uma ampla gama de disciplinas científicas e clínicas. Esta técnica é usada para examinar a presença, abundância relativa, massa molecular relativa e modificações pós-translacionais de proteínas1. Combinado com a análise digital de imagens, este método pode analisar de forma confiável a abundância de proteínas e modificações proteicas2. Embora a mancha ocidental seja realizada rotineiramente, é um método demorado e intensivo em mão-de-obra. São necessárias longas incubações do anticorpo com membranas. Aqui, descrevemos uma modificação do método de incubação que supera essa limitação sem sacrificar a sensibilidade.
Durante a incubação da membrana, os anticorpos flutuam em solução enquanto os antígenos são imobilizados na membrana. Devido à sua alta afinidade, a taxa de ligação de anticorpos ao antígeno é mais rápida do que a difusão de anticorpos da solução a granel para a membrana. Isso cria uma baixa concentração "camada de esgotamento"(Figura 1). Pode levar horas para anticorpos mais distantes chegarem à membrana por meio da difusão passiva, que é o principal fator responsável por longos tempos de incubação nesta técnica. Esse efeito é chamado de limitação de transporte de massa (MTL)3. Foi proposto que a drenagem repetitiva e a reposição da solução contendo anticorpos podem interromper a camada de esgotamento e superar o MTL4. Aqui, elaboramos uma técnica única que implementa este conceito de drenagem e reposição cíclica (CDR) para diminuir o efeito do MTL em um protocolo tradicional de manchas ocidentais e comentou encurtar o período de incubação necessário.
Para manter a sensibilidade de detecção com um curto tempo de incubação, fizemos uso de uma tecnologia de aprimoramento da imunoreaction que acelera a reação de anticorpos de antígeno, melhorando assim a relação sinal-ruído5. Muitos agentes de imunoreação disponíveis comercialmente (IRE) consistem em 2 componentes (Solução 1 e 2, ver Tabela de Materiais). A composição proprietária ou mecanismo de ação do IRE não foi divulgada, mas descobrimos anteriormente que o IRE diminuiu a constante de dissociação entre o antígeno e o anticorpo em ensaios de ligação de fase sólida, indicando que o aumento da afinidade é, pelo menos em parte, responsável pelo efeito de aumento do IRE6. A combinação de CDR com IRE produz um protocolo de manchas ocidentais de ultra-alta velocidade que reduz todo o tempo de procedimento sem sacrificar a sensibilidade.

<table><tbody><tr><td>14 mL Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap</td><td>Falcon</td><td>352059</td><td></td></tr><tr><td>Apollo Transparency Film for Laser Printers</td><td>N/A</td><td>N/A</td><td>Any kinds of Laser Printer Transparency Film are fine.</td></tr><tr><td>Azure Imaging System 600</td><td>Azure Biosystems</td><td>Azure 600</td><td>Any kind of Image Acquiring Sytem is fine for chemiluminescent and/or fluorescent detection.</td></tr><tr><td>Can Get Signal Immunoreaction Enhancer Solution</td><td>TOYOBO</td><td>NKB-101</td><td></td></tr><tr><td>CARNATION Instant Nonfat Dry Milk</td><td>Carnation</td><td>N/A</td><td></td></tr><tr><td>ECL anti-mouse IgG, Horseradish peroxidase linked F(ab&amp;rsquo;)2 fragment from sheep</td><td>GE Healthcare</td><td>NA9310V</td><td></td></tr><tr><td>ECL anti-rabbit IgG, Horseradish peroxidase linked F(ab&amp;rsquo;)2 fragment from donkey</td><td>GE Healthcare</td><td>NA9340V</td><td></td></tr><tr><td>HYBAID Micro-4</td><td>N/A</td><td>N/A</td><td>Any hybridization oven is fine as long as the tubes are rotated evenly at a horizontal position.</td></tr><tr><td>Immobilon-P PVDF Membrane, 0.45 &amp;micro;m pore size</td><td>Millipore Sigma</td><td>IPVH304F0</td><td></td></tr><tr><td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td><td>LICOR</td><td>926-68070</td><td></td></tr><tr><td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td><td>LICOR</td><td>926-32211</td><td></td></tr><tr><td>KPL LumiGLO Reserve Chemiluminescent Substrate</td><td>seracare</td><td>5430-0049</td><td></td></tr><tr><td>Mouse anti-&amp;szlig; actin antibody</td><td>Millipore Sigma</td><td>A5316</td><td></td></tr><tr><td>Odyssey Blocking Buffer (PBS)</td><td>LICOR</td><td>927-40100</td><td>Blocking Buffer for fluorescent detection</td></tr><tr><td>OXO Salad Spinner</td><td>OXO</td><td>32480V2B</td><td>Any salad spinner is fine as long as the PVDF membranes are rinsed vigorously without tear.</td></tr><tr><td>Parafilm Sealing Film</td><td>Bemis</td><td>Parafilm M PM996</td><td>semi-transparent flexible film</td></tr><tr><td>Polyethylene Flat-Top Screw Caps for 50 mL Conical Bottom Centrifuge Tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Rings to hold 14 ml tube in the center of 50 ml tube</td><td>N/A</td><td>N/A</td><td>Prepared in a machine shop</td></tr><tr><td>Rabbit anti-6X His tag antibody</td><td>Abcam</td><td>ab9108</td><td></td></tr><tr><td>Tween20</td><td>Millipore Sigma</td><td>P2287</td><td></td></tr></tbody></table>

ultra-high-speed western blot using immunoreaction enhancing technology

Uma mancha ocidental (também conhecida como imunoblot) é um método canônico para pesquisa biomédica. É comumente usado para determinar o tamanho relativo e abundância de proteínas específicas, bem como modificações de proteína pós-translacional. Esta técnica tem uma rica história e permanece em uso generalizado devido à sua simplicidade. No entanto, o procedimento de mancha ocidental leva horas, até dias, para ser concluído, com um gargalo crítico sendo os longos tempos de incubação que limitam seu rendimento. Estas etapas de incubação são necessárias devido à lenta difusão de anticorpos da solução a granel para os antígenos imobilizados na membrana: a concentração de anticorpos perto da membrana é muito menor do que a concentração a granel. Aqui, apresentamos uma inovação que reduz drasticamente esses intervalos de incubação, melhorando a ligação de antígeno através da drenagem cíclica e reposição (CDR) da solução de anticorpos. Também utilizamos uma tecnologia de aprimoramento da imunoreação para preservar a sensibilidade do ensaio. Uma combinação do método CDR com um agente de aumento de imunoreaction comercial aumentou o sinal de saída e reduziu substancialmente o tempo de incubação de anticorpos. A mancha ocidental de ultra-alta velocidade resultante pode ser realizada em 20 minutos sem qualquer perda de sensibilidade. Este método pode ser aplicado a manchas ocidentais usando a detecção de chemiluminescente e fluorescente. Este protocolo simples permite que os pesquisadores explorem melhor a análise da expressão proteica em muitas amostras.

Este exemplo ilustra a eficácia do método CDR juntamente com a tecnologia de imunoenhancing na mancha ocidental. A incubação estática foi realizada em uma película flexível semi-transparente onde as membranas PVDF foram incubadas com a solução de anticorpos, enquanto a incubação da CDR estava em um forno de hibridização por tubos rotativos que continham as membranas e a solução de anticorpos (Movie). A Figura 2 mostrou quantidades de antígeno e anticorpo, respectivamente, nec...

Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Uma técnica de mancha ocidental de ultra-alta velocidade é desenvolvida melhorando a cinética da ligação antígeno-anticorpo através da tecnologia de drenagem e reposição cíclica (CDR) em conjunto com um agente de aumento de imunoreaction.

Blot ocidental de alta velocidade usando tecnologia de aprimoramento de imunoreaction

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

ultra-high-speed-western-blot-using-immunoreaction-enhancing

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

5.8K visualizações.  National Institutes of Health. Uma técnica de mancha ocidental de ultra-alta velocidade é desenvolvida melhorando a cinética da ligação antígeno-anticorpo através da tecnologia de drenagem e reposição cíclica (CDR) em conjunto com um agente de aumento de imunoreaction.

Vídeo: Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Um protocolo otimizado para a mobilidade eletroforética ensaio de desvio usando oligonucleotídeos Infrared corante fluorescente-etiquetados

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Bioquímica

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

Western Blotting Usando o Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

Biologia

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Detecção altamente sensível e quantitativa de proteínas e suas isoformas pelo método isoeléctrica capilar

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Análise immunoblot

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western Blotting: Preparação de amostras para detecção

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

O mais rápido na Cidade Ocidental: um toque contemporâneo na Análise clássico Western Blot

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

A Guide to Modern quantitativa fluorescente Western Blotting com estratégias de resolução de problemas

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

Técnicas Baseadas em Imunoprecipitação: Purificação de Proteínas Endógenas Usando Esferas de Agarose

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

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Imunologia

The Western Blot

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Next, the membranes are probed with antibodies in a process called immunboblotting. Immunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. The detection of antibodies takes place using reporter systems which includes the use of enzymes. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called densitometry.
This video-article presents an overview of the western blot technique by describing western transfer, the use of antibody detection, and image analysis. The steps involved with western transfer such as the assembly of the transfer sandwich and transfer conditions are discussed in detail as well as the theory behind antibody binding and detection of those antibodies. The broad applications of this technique are described through several examples including the detection of protein-protein interactions and identification of individual proteins within protein complexes.

Western Blotting: Western Transfer, Antibody Detection, and Image Analysis

O Western Blot

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples.  Following separation by a technique ...

Biologia Básica

Métodos Básicos em Biologia Celular e Molecular

Chemiluminescent Western Blot Assay for Protein Processing

Source: Hillert-Richter, L. K. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62842">Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.</a> J. Vis. Exp. (2021).
This video describes the chemiluminescence-based western blotting of immunoprecipitated lysates. The technique helps determine protein processing and activation. The chemiluminescent signal detected is proportional to the level of protein processing during its activation.

Ensaio de Western Blot Quimioluminescente para Processamento de Proteínas

Source: Hillert-Richter, L. K. et al., Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex. J. ...