Esta pesquisa foi apoiada pelo Programa de Pesquisa Intramural do NIDCR.

1. Visão geral esquemática: Este vídeo mostra os passos envolvidos na realização do ensaio LIPS. Antígenos são expressos em células como Cos1 recombinante luciferase Renilla (Ruc) antigénio de fusões e extratos são obtidos e utilizados sem purificação. O ensaio LIPS é iniciada através da incubação crud e Ruc antígeno extratos com o soro do paciente em poços da microplaca. A mistura antígeno-anticorpo é então transferido para uma placa de 96 poços de filtro contendo proteína A / G grânulos para capturar moléculas de IgG. Depois de lavar a placa de filtro contendo a proteína Um anticorpo / G contas, ligado Ruc antígeno é medida pela adição de substrato celenterazina e unidades de luz são medidos com um luminómetro. PARTE 1: transfecção de plasmídeos para a produção de proteínas Renilla Antígeno-Fusion Set-up: Cos1 células são cultivadas em DMEM FCS-10%, utilizando protocolos padrão da cultura de tecidos. Plasmídeos para Renilla fusões luciferase tem sido descrito anteriormente 1. DNA para estes plasmídeos é preparado usando um kit da Qiagen Midiprep. O rendimento deve ser de aproximadamente 1 mg -3. Medir a concentração de DNA e armazenada como um mg 1000 / ml de solução a -20 ° C. Procedimento: <ol><li> Um dia antes de dividir transfecção, Cos-1 células em novo 100 x 20 mm em pratos de cerca de 2 X 10 6 por placa e incubar a 37 ° C. </li><li> No dia seguinte, o Cos-1 células deve ser 80-95% confluentes. Rótulo 1,5 ml tubos de polipropileno de microcentrífuga para cada DNA plasmídeo a ser transfectadas. Permitir que o reagente de transfecção FuGENE-6, que é armazenado a 4 ° C, para aquecer a temperatura ambiente. </li><li> Adicionar 94 mL de Opti-MEM mídia para cada tubo de microcentrífuga. Em seguida adicionar 6 mL de FuGENE 6 a mídia Opti-MEM sem tocar na parede lateral. </li><li> Incubar a mistura por 5 minutos em temperatura ambiente. </li><li> Adicione 1-2 mg (1mg/ml de ações DNA) de plasmídeo para a fusão do antígeno Renilla luciferase construir. Misturar e incubar a mistura por 15 minutos em temperatura ambiente. </li><li> Transferir o DNA FuGENE solução 6-Opti-MEM para as células por gotejamento é uniforme na mídia do Cos1 células. </li></ol> PARTE 2: A colheita Renilla antígeno-Fusions <ol><li> Dois dias após a transfecção, o Cos-1 as células são colhidas. Este é iniciado pela remoção da mídia e, em seguida, enxaguar as células com 6 ml de PBS. Após decantação da PBS, pipeta fora qualquer PBS residual do prato de cultura de tecidos. </li><li> Adicionar 1,4 ml de tampão de lise fria composta por 50 mM Tris, pH 7,5, 100 mM NaCl, 5 mM MgCl 2, 1% Triton X-100 glicerol, 50% e os inibidores da protease (2 comprimidos de coquetel completo miniprotease inibidor por 50 ml de tampão de lise). Células da colheita com uma scrapper de células e rápida de transferir metade do lisado de cada um dos dois tubos de 1,5 ml de microcentrífuga no gelo. </li><li> A Sonifier Branson 150 é usado para quebrar as células abertas. Coloque o tubo de microcentrífuga contendo o lisado celular no gelo e pulso durante 5 segundos, 5 segundos e 5 segundos com as configurações de sonicação de 2, 2 e 4, respectivamente. </li><li> Centrifugar o lisado celular a 12.500 RPM por duas rodadas 4 minutos a 4 ° C. Após a rodada em primeiro lugar, inverter cuidadosamente os tubos para remover os detritos frouxamente ligados a partir da parede lateral do tubo. Após a rodada em segundo lugar, transferir cuidadosamente o sobrenadante, sem perturbar o sedimento, a partir dos dois tubos para um tubo de microcentrífuga novo no gelo. </li><li> Cálculo das unidades de luz (CN) por mL de lisado. Para medir o LU, diluir 1 ml de lisado com 8 mL de PBS em um tubo de microcentrífuga novo. Adicionar diretamente os 100 l do substrato 1X celenterazina à mistura diluída e imediatamente luminescência medida no tubo com um luminômetro tubo (20/20 n Turner Scientific) com uma leitura de 5 segundos. </li><li> Armazenar o lisado Ruc antígeno a -20 ° C por 1-2 dias ou armazenar por longo período de tempo em alíquotas a -80 ° C. </li></ol> PARTE 3: Preparar uma Sera Placa Mestre <ol><li> Fazer um prato principal sera primeiramente adicionando 450 mL de tampão A (50 mM Tris, pH 7,5, 100 mM NaCl, 5 mM MgCl 2, 1% Triton X-100) em cada poço de uma microplaca de 96 poços profundos de polipropileno . Nesta etapa, a Red, corante Fenol, também podem ser incluídos em tampão A (concentração final é de 0,2 mg / ml em Tampão A) para atuar como um traçador para o monitoramento da adição da amostra futuro soros e outras medidas do ensaio LIPS. </li><li> Em seguida adicione 50 ul de soro de cada amostra aos diferentes poços contendo 450 mL de tampão A. Note que este é uma diluição de 1:10 dos soros em tampão A. Tipicamente soros não é adicionado aos dois últimos poços da placa de mestre, pois este é reservado para os espaços em branco buffer. </li><li> Antes de usar a placa de mestre, ele é amplamente abalado (1-2 horas) em uma plataforma rotador. O soro no prato principal é stasável por pelo menos 1 mês (ou mais) a 4 ° C, se armazenados corretamente para evitar a evaporação. </li><li> Como descrito abaixo, esta placa mestre fornece 10 ml de soro diluído para ser repetidamente removido para o perfil dos soros contra antígenos múltiplos. Mestre placas maiores e menores também podem ser empregadas. Selar a placa bem com Parafilm quando não estiver sendo usado. </li></ol> PARTE 4: ensaio LIPS <ol><li> Polipropileno de 96 placas de microtitulação bem rasas são usados ​​para testar soros. Na primeira etapa, adicionar 40 mL de tampão A para cada poço da placa de 96 poços usando uma micropipeta de 8 canais. </li><li> Em seguida, pegue 10 ml de soro diluído (1 equivalente soros mL) a partir da placa principal e adicioná-lo diretamente a cada poço da placa de trabalho usando uma micropipeta de 8 canais. </li><li> Um mix master contendo o extrato Ruc antígeno é o próximo formulado tal que 1 X 10 7 unidades de luz (LU) é adicionado em 50 mL de tampão A para cada poço. Entradas mais baixas (menos de 1 X 10 6) também pode ser usado, mas resultam em um menor intervalo dinâmico. Faça esta mistura mestre e pipeta mL 50 de Ruc antígeno-mistura para cada poço. </li><li> Incubar a placa em um agitador rotativo por 1 hora à temperatura ambiente. </li><li> Durante a incubação, adicionar 5 mL de uma suspensão de 30% do ULTRALINK proteína A / G contas (Pierce Biotecnologia, Rockford, IL) em PBS para o fundo de cada poço de uma nova de 96 poços filtro de placa HTS (Millipore, Bedford, MA) . </li><li> Após a incubação de 1 hora, transferir a 100 l Ruc antígeno-anticorpo mistura de reação para 96 ​​placas de filtro bem HTS contendo a proteína A / G contas usando uma micropipeta de 8 canais. </li><li> Incubar a placa de 96 poços filtro em um agitador rotativo por 1 hora em temperatura ambiente. </li><li> De seguida, lavar a placa de filtro em um distribuidor de vácuo. Cada poço é lavado oito vezes com 100 ul de tampão A, seguido por duas vezes com 100 ul de PBS. Isto pode ser feito manualmente ou com uma estação de trabalho de pipetagem robótico. </li><li> Após a última lavagem, o vácuo é desligado. Remova a placa de filtro e secar secar utilizando uma pilha de toalhas de papel ou papel-filtro certificando-se de remover a umidade na parte superior e inferior da placa. </li></ol> PARTE 5: Medição de análise de dados e Luminescência Set-up: A LB Berthold 960 Centro luminômetro é usado para determinar a luminescência em cada cavidade utilizando um injector único. Uma vez que a máquina está ligada, lavar o injetor com H 2 O destilada usando o ciclo de lavagem injector. Celenterazina substrato é preparado usando o kit Promega substrato Renilla como descrito pelo fabricante. Tipicamente ml 6 de mix 1X celenterazina substrato (ou seja, 60 mL de ações celenterazina mais 6 ml de 1X tampão) está preparada para aprontar a máquina e executando um prato cheio de 96 poços. Antes de analisar a placa, o LB Berthold 960 Centro luminômetro é preparado com 1X substrato celenterazina. Abrir um arquivo de programa que contém a definição para injetar o substrato e leitura da placa. Para estas medições, 50 ul de substrato celenterazina é injetado, a placa é abalada por 2 segundos, seguido por um 5 segundos de leitura de luminescência. <ol><li> Embora o luminômetro placa tem a capacidade de ler a placa inteira, uma leitura parcial da placa também pode ser selecionada (no menu de leitura). Iniciar o programa, que inicia a leitura da placa. </li><li> Após a corrida, retire a placa de filtro de microtitulação prontamente para impedir derrame para o luminômetro. Exportar os dados gerados com o programa MikroWin em um formato Excel para análise. </li><li> Recomendamos avaliar a soros de duas corridas independentes. Dados é, então, em média, para gerar os valores LU título para cada amostra. Estes valores podem ainda ser ajustado para subtrair os espaços em branco buffer. </li><li> Análise de dados adicionais, como determinar o corte pode ser calculada tomando a média mais 3 ou 5 desvios-padrão dos valores de controle. </li></ol>

<ol>
	<li>Burbelo, P. D., Goldman, R., Mattson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+immunoprecipitation+method+for+quantitatively+measuring+antibody+responses+in+clinical+sera+samples+by+using+mammalian-produced+Renilla+luciferase-antigen+fusion+proteins.">A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.</a> BMC Biotechnol. 5, 22-22 (2005).</li><li>Burbelo, P. D., Leahy, H. P., Iadarola, M. J., Nutman, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Four-Antigen+Mixture+for+Rapid+Assessment+of+Onchocerca+volvulus+Infection.">A Four-Antigen Mixture for Rapid Assessment of Onchocerca volvulus Infection.</a> PLoS Negl Trop Dis. 3, e438-e438 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+robust+luminescent+profiling+of+anti-La+and+other+autoantibodies+in+Sj+gren&#x2019;s+syndrome.">Sensitive and robust luminescent profiling of anti-La and other autoantibodies in Sj gren&#x2019;s syndrome.</a> Autoimmunity. 139, 241-245 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+antibody+quantification+and+generation+of+whole+proteome+antibody+response+profiles+using+LIPS+(luciferase+immunoprecipitation+systems).">Rapid antibody quantification and generation of whole proteome antibody response profiles using LIPS (luciferase immunoprecipitation systems).</a> Biochem Biophys Res Commun. 352, 889-895 (2007).</li><li>Burbelo, P. D., Groot, S., Dalakas, M. C., Iadarola, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+definition+profiling+of+autoantibodies+to+glutamic+acid+decarboxylases+GAD65/GAD67+in+stiff-person+syndrome.">High definition profiling of autoantibodies to glutamic acid decarboxylases GAD65/GAD67 in stiff-person syndrome.</a> Biochem Biophys Res Commun. 366, 1-7 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+luminescence+assay+for+autoantibodies+to+mammalian+cell-prepared+insulinoma-associated+protein+2.">A new luminescence assay for autoantibodies to mammalian cell-prepared insulinoma-associated protein 2.</a> Diabetes Care. 31, 1824-1826 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+diagnosis+of+human+herpes+simplex+virus+type+1+and+2+infections+by+luciferase+immunoprecipitation+system+assay.">Serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system assay.</a> Clin Vaccine Immunol. 16, 366-371 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+quantitative+serological+detection+of+anti-cytomegalovirus+(CMV)+antibodies.">Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies.</a> Virol J. 6, 45-45 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-antigen+mixture+containing+v-cyclin+for+serological+screening+of+human+herpesvirus+8+infection.">Four-antigen mixture containing v-cyclin for serological screening of human herpesvirus 8 infection.</a> Clin Vaccine Immunol. 16, 621-627 (2009).</li><li>Ramanathan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+luciferase+immunoprecipitation+systems+assay+enhances+the+sensitivity+and+specificity+of+diagnosis+of+Strongyloides+stercoralis+infection.">A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection.</a> J Infect Dis. 198, 444-451 (2008).</li></ol>

LIPS requer otimização de ensaio mínima e, devido à sua simplicidade, dados de alta qualidade pode ser tipicamente gerados usando LIPS em menos de duas semanas para qualquer dado antígeno. Os passos mais demorados são a clonagem e gerando o vetor de expressão apropriada plasmídeo contendo o antígeno de fusão Ruc. Uma vez que esses plasmídeos são gerados, antígenos únicos ou múltiplos podem ser rapidamente testado com LIPS como descrito acima. Deve-se observar que os lábios é bastante versátil de modo que sera também pode ser testada em um formato de tubo ou, alternativamente, em uma placa de microtitulação de filtro com o auxílio de uma estação de trabalho robótico BIOMEK 2 ou uma máquina de lavar prato com filtração a vácuo. Este sistema altamente escalável permite a LIPS paralelo perfil de um painel de auto-antígenos humanos 3 ou o proteoma inteiro de um vírus 4. É provável que muitos perfis diferentes de anticorpos gerados por LIPS será útil para o diagnóstico 3-9, antígeno descoberta 9, seguindo o curso do tratamento 10, o monitoramento da vacina e tem amplas implicações para os estados de doença específica e para a saúde pública em geral.

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>HiSpeed Plasmid Midi KIt</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
<tr>
<td>100 x 20 mm tissue culture dishes</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>353003</td>
<td>&nbsp;</td>
<tr>
<td>Opti-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>FuGENE 6</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1814443 </td>
<td>&nbsp;</td>
<tr>
<td>Complete, Mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836153001</td>
<td>&nbsp;</td>
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<td>Polypropylene 96 DeepWell 1 ml plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120 </td>
<td>Deep well plate with lid and seal with Parafilm</td>
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<td>Polypropylene microtiter plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120</td>
<td>&nbsp;</td>
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<td>HTS Filter plate</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSBVN1B50</td>
<td>&nbsp;</td>
<tr>
<td>Ultralink Immobilized Protein A/G</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>53133 (10ml)</td>
<td>&nbsp;</td>
<tr>
<td>Renilla Luciferase Assay System</td>
<td>&nbsp;</td>
<td>Promega </td>
<td>E2820</td>
<td>For 2000 assays</td>
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<td>Centro microplate luminometer</td>
<td>&nbsp;</td>
<td>Berthold</td>
<td>LB 960</td>
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antibody profiling by luciferase immunoprecipitation systems (lips)

Tecnologias para a compreensão abrangente e quantificar perfis de anticorpos para auto-antígenos e agentes infecciosos podem gerar novos insights sobre os mecanismos da doença e pode elucidar novos marcadores para substratify doença com diferentes características clínicas e entender melhor a patogênese. Nós desenvolvemos um método altamente quantitativa denominada luciferase Sistemas de imunoprecipitação (LIPS) para perfis respostas de anticorpos do paciente soros para auto-antígenos e antígenos de patógenos associados à infecção. Ao contrário dos ELISAs, o LIPS altamente sensível é facilmente implementada para pesquisa humoral perfis de resposta sorológica a antígenos diferentes em um formato universal e produz títulos de anticorpos dinâmica varia até 6 log<sub&gt; 10</sub&gt; Para alguns antígenos. Nestes estudos, perfis quantitativos por LIPS do paciente respostas humoral contra os painéis de antígenos ou até mesmo todo o proteoma de alguns patógenos (ie HIV), normalmente é mais informativa do que testar um único antígeno pelo método ELISA. Além disso, LIPS também elimina o tempo eo esforço necessário para produzir antígenos altamente purificados, bem como o trabalho intensivo passos necessários para a otimização do ensaio ELISA padrão. Aqui nós fornecemos um protocolo detalhado descrevendo os aspectos técnicos da realização de ensaios para LIPS prontamente profiling respostas de anticorpos contra antígenos únicos ou múltiplos.

Watch this Scientific Journal Video about Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS at JoVE.com

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

Os aspectos técnicos do desempenho LIPS (Sistemas de imunoprecipitação luciferase) são descritos. A abordagem global envolve expressar genes quiméricos codificando antígenos fundido a Renilla luciferase (Ruc) em células de mamíferos. Crude Ruc-antígeno extratos são então preparadas e, sem a purificação, empregada em ensaios de imunoprecipitação para quantificar anticorpos.

Perfil de anticorpos por sistemas de imunoprecipitação luciferase (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

antibody-profiling-by-luciferase-immunoprecipitation-systems-lips

Research

JoVE Journal

Biology

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

26.8K visualizações.  National Institute of Dental and Craniofacial Research, National Institutes of Health. Os aspectos técnicos do desempenho LIPS (Sistemas de imunoprecipitação luciferase) são descritos. A abordagem global envolve expressar genes quiméricos codificando antígenos fundido a Renilla luciferase (Ruc) em células de mamíferos. Crude Ruc-antígeno extratos são então preparadas e, sem a purificação, empregada em ensaios de imunoprecipitação para quantificar anticorpos.

Vídeo: Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

Fenômenos AC eletrocinéticas Gerado por Estruturas microeletrodos

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

Biologia

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Imunoprecipitação DNA metilado

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

FCM-IP: Imunoprecipitação Detectado por citometria de fluxo

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Transcrição in vitro e nivelamento de Gaussia mRNA luciferase Seguido por HeLa transfecção celular

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Microarray de anticorpos quimicamente bloqueada para Multiplexed perfil de alta capacidade de glicosilação proteína específica em amostras complexas

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Análise da Tradução Iniciação Durante condições de estresse por polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Profiling Individual células estaminais embrionárias humanas por RT-PCR quantitativo

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Avaliação da Seletiva mRNA Tradução em células de mamíferos por polissoma Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...