Somos gratos a Michela de Martino, Wilfred R. Hagen, e Kourosh Honarmand Ebrahimi para discussões úteis. Este trabalho foi apoiado por fundos start-up da Universidade Delft de Tecnologia.

1. Dps Expressão e purificação Obtenção de proteína de alta pureza é um primeiro passo essencial para o ensaio de protecção de ADN. A purificação da proteína pode ser realizada Dps em 4 a 5 dias. <ol><li> Transformar uma estirpe de protease deficiente de E. coli (tal como BL21 (DE3) pLysS), com um vector pET (por exemplo, pET17) na qual a sequência de codificação de proteína Dps foi clonado. </li><li> Streak as células transformadas para fora em Caldo (LB) ágar Luria contendo antibióticos apropriados (como ampicilina e cloranfenicol para os exemplos fornecidos em 1.1). Incubar as placas durante a noite a 37 ° C. </li><li> Inocule uma colónia isolada a partir da placa em 30 ml de meio LB contendo antibióticos apropriados. Incubar durante a noite a 37 ° C enquanto agitando a 200-250 rpm. </li><li> Inocular 2 x 1 L de meio LB contendo antibióticos adequados com 10 ml cada uma da cultura durante a noite. Crescer a 37 ° C, sob agitação, a DO600 </sub&gt; de aproximadamente 0,6. (Cultivo de células mais elevada aumenta o rendimento de proteína OD, sensivelmente sem comprometer a pureza do produto final.) Induzir a expressão de Dps pela adição de IPTG para uma concentração de 0,3 mM e incuba-se a 37 ° C durante 3-4 horas, com agitação. </li><li> Colheita de células por centrifugação a 6000 xg durante 15 min. Voltar a suspender as pelotas de células em 7,5 ml de DEAE tampão A (50 mM HEPES-KOH, pH 7,5, NaCl 100 mM, EDTA 0,1 mM) por L de cultura celular induzida. As amostras podem ser congeladas em azoto líquido e armazenadas a -80 ° C, se se desejar. O processo pode então ser continuada por descongelamento das amostras num banho de água a 4 ° C. </li><li> Adicionar uma mistura de inibidores de protease (tais como os inibidores da protease Calbiochem Conjunto III, em 0.167 mL por ml de suspensão de células) para a suspensão de células para evitar a degradação de Dps overexpressed. </li><li> Prepare extrato livre de células utilizando uma prensa francesa. Primeiro a imprensa francesa, com 20 ml DEAE tampão A (se estiver usando um modelo de fluxo contínuo), então disrupt a amostra duas vezes a 20 kpsi. Centrifuga-se o ligado a 30.000 x g durante 35 min a 4 ° C para clarificar as partículas insolúveis, e guardar o sobrenadante. O sobrenadante pode então ser congelados usando azoto líquido e armazenadas a -80 ° C, se se desejar. </li><li> Equilibrar a coluna de 30 ml de DEAE Sepharose CL-6B DEAE com Tampão A, utilizando um FPLC. Executar tampão através da coluna a 1-2 ml / min, com uma pressão máxima de 0,2 MPa ao longo do fundo. Carregar o extracto isento de células para a coluna, e começa a receber o fluxo através de uma vez que o sinal de OD 280 começa a subir acima da linha de base. Lava-se a coluna com DEAE Tampão A até que o valor de DO 280 retorna à linha de base, enquanto continuamente recolhendo o escoamento. O fluxo através deve conter a maior parte da proteína de Dps, isenta de ADN ligado. Lava-se a coluna com 100% de tampão B (50 mM HEPES-KOH, pH 7,5, NaCl 1 M, EDTA 0,1 mM). Este fluido contém complexos Dps-DNA, bem como outras proteínas e pode ser descartada. </li><li> Remover anúnciocional proteínas contaminantes através de precipitação com sulfato de amónio. Determinar o volume de fluxo através reunidas. Lentamente (durante um período de 10-20 minutos) adicionar sulfato de amónio de pequeno grão seco a 62% de saturação (390 mg por ml de solução), a 4 ° C, enquanto se agitava bem. Agita-se a mistura durante um adicional de 20-30 minutos após a adição da última porção de sulfato de amónio para assegurar o equilíbrio completo. Dps permanecerá solúvel, enquanto que as proteínas contaminantes irá precipitar para fora da solução. </li><li> Remover as proteínas precipitadas por centrifugação a 20.000 xg durante 30 min a 4 ° C e guardar o sobrenadante. </li><li> Lentamente, adicionar uma mg de sulfato de amónio adicional por 227 ml de sobrenadante para atingir 94% de saturação e agitar durante 20-30 minutos para garantir o equilíbrio completo. Dps precipita nesta concentração elevada de sal e pode ser recolhido por centrifugação a 20.000 xg durante 30 min a 4 ° C. Dps estará na pelete eo sobrenadante pode ser descartado. O sedimento pode ser armazenada a -80 ° C, se desired. </li><li> Ressuspender o DPS contendo sedimento em tampão de ressuspensão (50 mM de HEPES-KOH, pH 7,5, NaCl 150 mM, EDTA 0,1 mM). Ajustar o volume da amostra de 2,5 ml. </li><li> Tampão de troca para remover o sulfato de amónio, utilizando uma coluna de filtração em gel PD-10. Equilibrar o leito de gel com 25 ml de tampão de ressuspensão. Aplique a 2,5 ml de amostra Dps para o topo da coluna PD-10 e deixe de molho dentro Descarte o flow-through. Recolhe Dps por eluição com 3,5 ml de tampão de ressuspensão. </li><li> Centrifuga-se o tampão permutado amostra a 16.000 xg durante 10 min a 4 ° C para remover todos os componentes insolúveis, e diluir com 6-10 ml de tampão de diluição (50 mM HEPES-KOH, pH 7,5, EDTA 0,1 mM), para assegurar que o concentração de sal é baixo o suficiente para Dps de se ligar à coluna Sepharose SP. Particularmente as amostras concentradas de Dps pode tornar-se menos solúvel por diluição para um tampão de menor sal, como evidenciado pela turvação do líquido, mas pode ser completamente ressolubilizado por meio da adição de uma pequena amount de solução de NaCl a 5 M (um adicional de 10-20 mM). </li><li> Equilibrar a coluna de fluxo de 30 ml de SP Sepharose Fast Food SP com tampão A (50 mM HEPES-KOH, pH 7,5, NaCl 50 mM, EDTA 0,1 mM). Executar tampão através da coluna a 1,5-2 ml / min, com um limite de pressão de 0,3 MPa ao longo do fundo. Carregar a amostra sobre a coluna. Lavar com SP tampão A até o OD 280 trace retorna à linha de base, e descartar o flow-through. Executar um gradiente linear de 150 ml de 0 a 100% de tampão B, enquanto recolhendo fracções de 2 ml. Dps vai eluir em um pico acentuado em torno de 50% B, embora a variação de 10% é possível. </li><li> Continuar as fracções de eluição em um gel SDS-PAGE 15%, e para verificar a contaminação de ADN medindo a razão OD 260 / OD 280 utilizando um espectrofotómetro. O gel só deve mostrar uma banda em torno de 19 kDa, eo OD 260 / OD 280 é normalmente em torno de 0,7. Reunir as frações mais puras PD. Usar uma unidade de filtro de centrífuga (tais como a unidade de ultrafiltração Amicon), com uma10K de peso molecular cut-off para concentrar as Dps e trocá-lo para um tampão de armazenamento (50 mM HEPES-KOH, pH 7,5, NaCl 50 mM). Alíquota da Dps purificados e congelamento em nitrogênio líquido para armazenamento a -80 ° C. </li></ol> 2. Protection Assay ADN O ensaio envolve várias etapas sensíveis ao tempo e deve ser cronometrado para o segundo para obter resultados reprodutíveis. Muitos ingredientes e passos de pipetagem estão envolvidos para uma mesa de pipetagem (ver Tabela 1), é aconselhável manter o controle de etapas e volumes. O tempo total necessário para o ensaio é de não mais do que 3 horas. <ol><li> Medir 30 ml de água em um tubo vedado; descarregar a água, com N2 durante 10 min (utilizando duas agulhas de seringa, uma no líquido, um acima dela) para remover o oxigénio a partir da solução. Adicionar 0,0168 g de FeSO 4 • 7H 2 O para a água para obter uma solução de estoque de 2 mM. Selar o frasco e lave com nitrogênio por 5 min mais, em seguida, misture. A solução deve serclaro, se qualquer indício de amarelo é detectável, preparar uma nova solução de ferro. </li><li> Adicione uma solução 100 mM de H 2 O 2 (10,87 mL de 9.2MH 2 O 2 em 1 ml de água), manter em gelo e protegida da luz por envolvimento em folha de alumínio. Utilize apenas por cerca de 1-2 horas após a preparação devido à deterioração espontânea. O estoque de peróxido de hidrogénio está igualmente sujeita a desagregação, que pode ser parcialmente melhorada por armazenamento adequada (no escuro, a 4 ° C). Controlo regular da concentração acionário, medindo os 240 OD são recomendados. </li><li> Descongelar uma alíquota de Dps rapidamente purificada num banho de água à temperatura ambiente, e manter em gelo. Centrifugar por 10 segundos a 4.000 xg em um table top picocentrifuge aos agregados precipitar. Medir a concentração após a centrifugação utilizando um espectrofotómetro (OD 280 = 1,547 indica uma concentração de 100 uM de monómeros Dps). </li><li> Diluir DNA linear de estoque-alta concentração usando tampão de reação 12x (1M de MOPS-KOH pH 7,0, 1 M de NaCl) para uma concentração de 100 ng / mL. ADN linear é usada para tornar mais fácil quantificação, mas o DNA de plasmídeo pode ser utilizado também. </li><li> Pipetar 1 mL da mistura de ADN-tampão para um tubo de PCR. Adicionar água suficiente para a reacção de modo que o volume final da reacção (menos de SDS) será de 12 ul. Este valor deve ser calculado com antecedência usando a tabela pipetagem. Adicionar Dps a uma concentração de 3 uM dodecamer final. Incubar durante 15 minutos à temperatura ambiente para permitir Dps para se ligar ao DNA. </li><li> Adicionar suficiente solução 2 mM de FeSO4 para atingir a concentração desejada. Uma experiência típica utiliza uma gama de concentrações de 0 a 1 mM de FeSO4. Concentrações mais elevadas irão causar mais extensa degradação de DNA, mas pode também levar à formação de férrico precipita na mistura de reacção. Rapidamente adicionar 1 mL da solução de 100 mM de H 2 O 2 (a uma concentração final de 10 mM), e incubar à temperatura ambiente durante 5 min para permitir que a FentonReação de degradação mediada por acontecer. </li><li> Adicionar 0,8 mL de 20% de SDS (dodecilsulfato de sódio). Misturar e incubar a 85 ° C durante 5 minutos para interromper os complexos de ADN-Dps. A SDS desestabiliza o complexo Dps-DNA e evita deslocamentos indesejados gel para o DNA, o que melhora a facilidade de quantificação. Um passo opcional é parar a reacção por cataliticamente degradar o peróxido de hidrogénio em produtos não-tóxicos. Para as condições descritas no presente protocolo, este passo não é necessária para obter uma ampla gama de degradação de ADN. </li><li> Incubar em gelo durante 1 min, adicionar corante de carga e carregar num gel de agarose. Executar o gel, e mancha de ADN, utilizando brometo de etídio. O gel deve ser coradas após electroforese, em vez de antes da electroforese, para evitar a SDS de interferir com a distribuição do brometo de etídio no gel. </li></ol>

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Não temos nada a divulgar.

O processo de purificação de Dps, conforme descrito neste artigo é muito robusto. Pureza tem sido consistentemente alta (&gt; 99%), não aparecem outras proteínas em gel de SDS-PAGE como bandas visíveis. Apesar disso, alguns lotes de Dps purificados parecem ter actividade de nuclease, tal como evidenciado pela degradação parcial de ADN quando incubadas com concentrações muito elevadas de Dps. Isto pode indicar a presença de ADNases altamente activos em baixa concentração que não fomos capazes de remover por...

Os organismos aeróbios deve constantemente competir com as espécies de oxigénio reactivas que podem danificar o DNA, bem como outras macromoléculas biológicas importantes. Uma ferramenta potente para neutralizar os efeitos tóxicos do dano oxidativo é a proteína de ligação de ADN a partir de células de privação (SAD). Desde a sua descoberta, em 1992, a partir de fome E. coli, uma cultura, Dps foi identificado em mais de 300 espécies de bactérias e 2 arqueobactérias. Regulação positiva maciça de Dps durante a fase estacionária torna a proteína nucleoid associada mais alta expressão de E. coli em condições de fome 3, 4. Além disso, tem sido mostrado Dps para preservar tanto a viabilidade bacteriana e da integridade do ADN durante muitos várias tensões, incluindo a fome, a elevada concentração de ferro, a exposição à luz UV, choque térmico, e do stress oxidativo 5, 6. Estruturalmente, Dps de auto-associados em um complexo estável homo-oligoméricas de 12 monômeros, which montar em uma concha vazia esférica. A ~ 4,5 nm de largura cavidade interna é acessível ao solvente através dos poros, que permitem a passagem de moléculas pequenas 7 o exterior, e que pode sequestrar metais, tais como ferro mineralizados 8. O efeito protetor do Dps deriva das suas diversas atividades bioquímicas, que incluem DNA não específico de ligação 1, atividade ferroxidase e armazenamento de ferro 8. Estudo detalhado das atividades bioquímicas benéficas Dps requer primeiro a sua purificação. Dps purificação é um procedimento elaborado, como PD devem ser separadas não apenas a partir de outras proteínas, mas também a partir de qualquer DNA ligado 7. O nosso processo de purificação optimizado utiliza várias técnicas comuns, que consiste em duas colunas de permuta iónica e um passo de precipitação com sulfato de amónio. Várias trocas de buffer são necessários, como Dps altamente concentrado pode precipitar para fora da solução em condições de pouca sal. Uma vez que a proteína foi purificada Dps, Pode ser aplicada a ensaios que medem directamente a sua actividade ferroxidase 8, estequiometria de ligação de ADN 9, e os mecanismos de ligação do ferro 10. Purificados Dps também tem outras aplicações potenciais. A estrutura esférica oca estável de Dps tem sido utilizado como andaime para armazenar partículas hidrofóbicas da proteína dentro da cavidade 11 e mesmo como uma câmara de reacção para sintetizar novas nanopartículas magnéticas 12. A capacidade de proteção de Dps para mediar danos devido a espécies reativas de oxigênio pode ser clara e diretamente demonstrado usando o ensaio de proteção DNA 13, 14. Neste procedimento in vitro, as espécies de radicais são produzidos quando o ferro catalisa H 2 O 2 a degradação química de Fenton. Estes radicais danificar directamente o ADN presente na reacção e pode degradar completamente em concentrações elevadas. Duas atividades principais Dps pode tanto contrariar diretamente os efeitos da Fenta produção do radical a-mediada. Dps reduz a concentração de ferro catalítico da mineralização, o consumo do peróxido de hidrogénio disponível no processo. Além disso, a ligação ao DNA Dps potencialmente pode protegê-lo fisicamente dos danos dos radicais e condensa-lo em um volume menor com menos área de superfície reativa. A combinação destas duas propriedades faz com que o ensaio de protecção de DNA adequado para a finalidade de medir a actividade protectora Dps. O ensaio de protecção de ADN é bastante versátil e pode ser usado para uma variedade de aplicações para além Dps caracterização. Os danos dos radicais é uma forma comum de estresse em células, e muitas proteínas e substâncias químicas diferentes são usados ​​para combatê-la. O princípio geral do ensaio, utilizando-se a integridade do DNA como um marcador para os danos dos radicais, pode ser usado em combinação com quase qualquer reacção de produção de radicais livres ou agente de neutralização. Entre outros, o ensaio tem sido usado com sucesso para determinar as propriedades anti-oxidantesde K. extrato paniculata para uso na indústria de alimentos 15, para caracterizar os efeitos do ácido úrico em hidroxila mediação dano 16, e para ganhar novos insights sobre a função da pele proteínas reguladoras da transcrição 17. Apesar dos inúmeros usos do ensaio nos trabalhos publicados, descobrimos que muitos optimização e foram necessários passos de resolução, o que torna a criação de ensaio pela primeira vez um processo desnecessariamente trabalhoso para muitos investigadores. O protocolo que apresentamos neste artigo tem como objetivo eliminar essa barreira para a entrada.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 </tr>
 <tr>
 <td>BL21(DE3)pLysS competent cells</td>
 <td>Promega</td>
 <td>L1195</td>
 </tr>
 <tr>
 <td>pET-17b DNA</td>
 <td>EMD-Millipore</td>
 <td>69663-3</td>
 </tr>
 <tr>
 <td>LB broth powder</td>
 <td>Sigma-Aldrich</td>
 <td>L3022</td>
 </tr>
 <tr>
 <td>Ampicillin sodium salt</td>
 <td>Sigma-Aldrich</td>
 <td>A0166</td>
 </tr>
 <tr>
 <td>Chloramphenicol</td>
 <td>Sigma-Aldrich</td>
 <td>C0378</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Sigma-Aldrich</td>
 <td>I6758</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>BDH</td>
 <td>441476L</td>
 </tr>
 <tr>
 <td>Potassium hydroxide</td>
 <td>Merck</td>
 <td>105033</td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>VWR</td>
 <td>443824T</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>E9884</td>
 </tr>
 <tr>
 <td>Protease Inhibitor Cocktail Set III</td>
 <td>EMD-Millipore</td>
 <td>539134</td>
 </tr>
 <tr>
 <td>DEAE-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>DFF100</td>
 </tr>
 <tr>
 <td>Ammonium sulphate</td>
 <td>Sigma-Aldrich</td>
 <td>A4418</td>
 </tr>
 <tr>
 <td>PD-10 Desalting Columns</td>
 <td>GE Healthcare LS</td>
 <td>17-0851-01</td>
 </tr>
 <tr>
 <td>SP-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>S1799</td>
 </tr>
 <tr>
 <td>Amicon Ultra Centr. Filter (10K MWCO)</td>
 <td>Millipore</td>
 <td>UFC901024</td>
 </tr>
 <tr>
 <td>Ferrous Sulphate heptahydrate</td>
 <td>Sigma-Aldrich</td>
 <td>F8048</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide solution</td>
 <td>Sigma-Aldrich</td>
 <td>216763</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Calbiochem</td>
 <td>475898</td>
 </tr>
 <tr>
 <td>SDS solution</td>
 <td>Bio-Rad</td>
 <td>161-0418</td>
 </tr>
 <tr>
 <td>Ethidium bromide</td>
 <td>Sigma-Aldrich</td>
 <td>E1510</td>
 </tr>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Static incubator</td>
 <td>Hettich</td>
 <td>INE500</td>
 </tr>
 <tr>
 <td>Shaking Incubator </td>
 <td>New Brunswick Sc.</td>
 <td>Inova 44</td>
 </tr>
 <tr>
 <td>Cooled centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Avanti J-E</td>
 </tr>
 <tr>
 <td>Table-top centrifuge</td>
 <td>Eppendorf</td>
 <td>5424</td>
 </tr>
 <tr>
 <td>Cell disrupter</td>
 <td>Constant Systems Ltd.</td>
 <td>TS2/40/AA/AA</td>
 </tr>
 <tr>
 <td>FPLC Purifier</td>
 <td>General Electric</td>
 <td>AKTA</td>
 </tr>
 <tr>
 <td>Airtight vials</td>
 <td>Cole-Parmer</td>
 <td>EW-08918-85</td>
 </tr>
 <tr>
 <td>Syringe needles</td>
 <td>BD</td>
 <td>305128</td>
 </tr>
 <tr>
 <td>Pipettes</td>
 <td>Eppendorf</td>
 <td>Z683779-1EA, Z683795-1EA</td>
 </tr>
 </tbody>
</table>

application of an in vitro dna protection assay to visualize stress mediation properties of the dps protein

O estresse oxidativo é um subproduto inevitável da vida aeróbica. Oxigênio molecular é essencial para o metabolismo terrestre, mas também participa em muitas reações prejudiciais dentro de organismos vivos. A combinação do metabolismo aeróbio e de ferro, que é outro composto vital para a vida, é suficiente para produzir radicais com a química de Fenton e degradar os componentes celulares. Degradação do DNA é sem dúvida o processo mais prejudicial envolvendo radicais intracelulares, como reparo de DNA está longe de ser trivial. O ensaio apresentado neste artigo oferece uma técnica quantitativa para medir e visualizar o efeito de as moléculas e enzimas sobre os danos de DNA mediada por radicais. O ensaio de proteção DNA é uma ferramenta simples, rápido e robusto para a caracterização in vitro das propriedades protetoras das proteínas ou produtos químicos. Isto envolve a exposição do ADN a uma reacção de oxidação prejudicial e adicionando várias concentrações do composto de interesse. A redução ou o aumento de danos no ADN, como uma função da concentração do composto é então visualizada utilizando electroforese em gel. Neste artigo, demonstrar a técnica do ensaio de protecção de DNA medindo as propriedades de protecção da proteína de ligação de ADN a partir de células de privação (SAD). Dps é uma mini-ferritina, que é utilizado por mais de 300 espécies bacterianas para combater poderosamente agressões ambientais. Aqui apresentamos o protocolo de purificação Dps e as condições de ensaio optimizado para avaliar a proteção do DNA por Dps.

O processo de purificação para Dps descritos aqui é muito reprodutível. Dps de purificação de acordo com o protocolo descrito, utilizando-se 2 L de E. cultura coli como um ponto de partida, normalmente produzir 2,5 ml de proteína contendo Dps 12 em concentrações entre 5 e 12 mM. Tempos de indução mais longas (4 horas) parecem reduzir essa variabilidade. Pureza da proteína é consistentemente superior a 99%, como evidenciado por SDS-PAGE, os géis (Figura 1). O n?...

Watch this Scientific Journal Video about Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein at JoVE.com

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

A proteína de ligação ao DNA de células famintas (SAD) desempenha um papel crucial no combate ao stress bacteriana. Este artigo discute a purificação da<em&gt; E. coli</em&gt; Dps eo protocolo para um<em&gt; In vitro</em&gt; Ensaio demonstrando proteção Dps mediada por DNA da degradação pelas espécies reativas de oxigênio.

A aplicação de um<em&gt; In vitro</em&gt; DNA Assay Proteção ao Visualize Propriedades Mediação stress do Dps Protein

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

application-an-vitro-dna-protection-assay-to-visualize-stress

Research

JoVE Journal

Biology

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

11.1K visualizações.  Delft University of Technology. A proteína de ligação ao DNA de células famintas (SAD) desempenha um papel crucial no combate ao stress bacteriana. Este artigo discute a purificação da E. coli Dps eo protocolo para um In vitro Ensaio demonstrando proteção Dps mediada por DNA da degradação pelas espécies reativas de oxigênio.

Vídeo: Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Um In vitro FluoroBlok Assay invasão tumoral

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Biologia

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.

Aplicação de Métodos Stopped fluxo de Cinética para investigar o mecanismo de ação de uma proteína de reparo do DNA

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Ensaios biofísicos para investigar as propriedades mecânicas do núcleo da célula Interphase: Aplicação de substrato Strain e Manipulação de microagulhas

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

A metilação in vitro Ensaio de Proteína Estudo Arginina metilação

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Ensaio Cometa como uma medida indireta do Systemic Estresse Oxidativo

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Medindo danos DNA e reparar no mouse Esplenócitos Depois crônica<em&gt; In Vivo</em&gt; A exposição a doses muito baixas de beta e gama-radiação

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Ensaio para imunoprecipitação da cromatina a identificação de Arabidopsis interações proteína-ADN<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

A Assembleia e Aplicação de &#39;tesoura Anéis&#39;: A endotelial novo modelo de Orbital, unidirecional e periódica fluxo de fluido e tensão de cisalhamento

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

Observação e quantificação do comportamento de acasalamento no nemátodo do pinheiro,<em&gt; Bursaphelenchus xylophilus</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...