O trabalho neste manuscrito foi apoiada por fundos do Departamento de Radioterapia Oncologia e Huntsman Cancer Institute e do Instituto Nacional de concessão de Saúde (R01-CA188520) para SB. Agradeço Danielle Johnson e Steven Bennett no meu laboratório para demonstrar o protocolo e explicando os seus benefícios. Sou grato ao Dr. Mahesh Chandrasekharan (Huntsman Cancer Institute) para comentários críticos sobre o manuscrito.

1. A cromatina imunoprecipitação (CHIP) seguinte BrdU Labeling <ol><li> Culturas de células NIH3T3 na presença de DMSO ou inibidores selectivos HDAC1,2-6, como descrito anteriormente. </li><li> A seguir ao tratamento com inibidores selectivos de DMSO ou HDAC1,2, adicionar BrdU para as células de uma cultura de tecido estéril capa. Incubar 2 x 10 6 NIH3T3 células plaqueadas em 10 ml de meio NIH3T3 com 20 uM de concentração final de bromodesoxiuridina (BrdU) durante 60 min num estéril 37 ° C incubadora de cultura de tecidos. </li><li> Proteínas intracelulares Crosslink ao DNA por adição de formaldeído directamente ao meio de cultura a uma concentração final de 1%. Incubar 2 X 10 6 células NIH3T3 com formaldeído à temperatura ambiente durante 10 minutos num agitador. </li><li> Extingue-se a ligação cruzada pela adição de glicina para uma concentração final de 125 mM. Incubar à temperatura ambiente durante 5 min num agitador. </li><li> Remover os meios de comunicação e recolher células em PBS num tubo de centrífuga de 15 ml. Spin cells a 956 xg durante 5 min a 4 ° C. Lava-se a pelete de células duas vezes com PBS gelado. Remover o PBS do tubo. Armazenar o sedimento celular reticulada sem PBS (o sobrenadante) a -80 ° C até à sua utilização. </li><li> Prepara-se o lisado a partir do sedimento celular reticulada por adição de tampão de FA140 500 ul (50 mM de HEPES KOH pH 7,5, 140 mM de NaCl, 1 mM de EDTA (pH 8,0), 1% de Triton-X-100 e desoxicolato de sódio a 0,1%) suplementado com cocktail inibidor de protease. </li><li> Distorcer a cromatina por sonicando as amostras de cinco vezes a 35% de energia e com um pulso de 15 segundos, 0,9 segundos em 0,1 segundos e fora configuração. Manter os tubos em gelo após cada rodada de sonicação para evitar o aquecimento das amostras. <ol><li> Verificar o corte num gel de ADN como a eficiência de cisalhamento vai variar entre diferentes sonicadores. Para verificar ultra-sons, reverter ligações cruzadas pela adição de NaCl a uma concentração de 0,16 M e executar etapas 1.17.1. - 1.22. Run eluída 10 ul DNA em gel de agarose 1,5% para verificar se o corte éentre 300-700 pb com uma intensidade média de 500 pb. </li></ol></li><li> Após sonicação, amostras de centrifugação durante 10 min a 17.949 xg, a 4 ° C, e transferir o sobrenadante para um novo tubo. <ol><li> Executar a estimativa de proteína utilizando o kit de ensaio de proteínas comerciais de acordo com o protocolo do fabricante. Use igual quantidade de lisado para Chip de controle e amostras experimentais. De um modo geral, usar 1 mg de lisado total por reacção de chip. </li></ol></li><li> Preparar pasta a 50% de proteína A-agarose usando tampão contendo FA140 cocktail inibidor de protease. Adicionar 1/10 volume de cocktail inibidor de protease. Calcular o volume de grânulos necessário com base no número de amostras no total. <ol><li> Lavar os grânulos de proteína A-agarose com tampão FA140 duas vezes para remover o tampão de armazenamento e adicionar um volume igual de tampão FA140 aos grânulos para fazer pasta a 50%. </li><li> Para remover as proteínas que se ligam de forma não específica às pérolas, adicionar 50 ul de pasta a 50% de Proteína A-agarose e emCubate as amostras a 4 ° C com rotação constante extremidade-sobre-extremidade num misturador espeto rotativo durante 30 min. </li></ol></li><li> Rotação das amostras a 956 xg, a 4 ° C durante um minuto. Recolhe-se o sobrenadante e realizar a imunoprecipitação pela adição de 8 pi de anticorpo quer H4K16ac ou 5 ul (5 ug) de 1 mg / ml de anticorpo SMARCA5 para o sobrenadante. Incubar a 4 ° CO / N com uma rotação de extremidade sobre extremidade constante num misturador de assador. </li><li> Salve o 1 / 10th do extrato para o controle &#39;input&#39; e configure-o como de lado a -20 ° C. A entrada tem de ser inversa reticulada juntamente com as amostras IP. </li><li> Usar uma quantidade igual de IgG de coelho (5 ul de 1 mg / ml) tal como a amostra de controlo negativo imunoprecipitação. </li><li> No dia seguinte, adicionar 50 ul de 50% de proteína A-agarose slurry ao extracto e incubar durante 1 hora a 4 ° C com rotação. </li><li> Pellet pérolas de agarose por centrifugação a 956 xg durante 2 min a 4 ° C, remover cuidadosamente o sobrenadante e wash os grânulos com os seguintes tampões sequencialmente. Em preparações de buffer, adicione os inibidores da protease direita antes de usar. <ol><li> Lavar com Baixo Sal complexo imune tampão de lavagem (FA140; concentração final de 50 mM de HEPES KOH pH 7,5, 140 mM de NaCl, 1 mM de EDTA (pH 8,0), 1% de Triton-X-100 e 0,1% de desoxicolato de sódio) durante 5 min a 4 ° C com rotação de extremidade sobre extremidade. </li><li> Lavar com alto de sal complexo imune tampão de lavagem (FA500; concentração final de 50 mM de HEPES KOH pH 7,5, 500 mM de NaCl, 1 mM de EDTA (pH 8,0), 1% de Triton-X-100 e 0,1% de desoxicolato de sódio) durante 5 min a 4 ° C com rotação de extremidade sobre extremidade. </li><li> Lava-se com LiCl complexo imune tampão de lavagem (concentração final de 10 mM de Tris-Cl pH 8,0, 250 mM de LiCl, 0,5% NP40 e 0,5% de desoxicolato de sódio) durante 5 min a 4 ° C com rotação de extremidade sobre extremidade. </li></ol></li><li> Após lavagens, tal como descrito acima, adicionar 200 ul de solução de eluição (concentração final de SDS a 1% e bicarbonato de sódio 100 mM) e incubate à temperatura ambiente durante 15 minutos com rotação de extremidade sobre extremidade à TA. </li><li> Rotação das amostras a 956 xg à temperatura ambiente e transferir o eluato para um novo tubo de 1,5 mL. Repetir o passo 1.15 com solução de eluição adicional de 200 ul. </li><li> A 400 ul do eluato, adicionar NaCl a uma concentração de 0,16 M e misturar bem. <ol><li> Além disso, adicionar solução de eluição de 400 uL a 10 uL de entrada que foi posto de lado (passo 1.11) e adicionar a uma concentração de NaCl de 0,16 M a inverter amostras de entrada de reticulação bem. Inverter a ligação cruzada das amostras por incubação a 65 ° C em banho de água durante 5 horas. </li></ol></li><li> Adicione 1 ml de EtOH a 100% para o produto de eluição inversa reticulado. Misturar bem e incubar à temperatura de -80 ° C durante 2 - 3 h a -20 ° ou CO / N. Rodar as amostras a 17.949 xg durante 15 min e descartar etanol. </li><li> Adicionar 800 ul de etanol a 70% para lavar o sedimento. Rotação em 17.949 xg durante 15 min a 4 ° C e descartar etanol. Rotação brevemente para remover o etanol residual qualquer. Secar as amostras a 37 ° C durante 10 min para remover o etanol residual. </li><li> Dissolve-se o ADN em 90 ul de ARNase e ADNase isenta de água e adiciona-se 2 ul de 10 mg / ml de RNase com uma solução a 0,2 mg / ml de concentração final. Incubar a 37 ° C em banho-maria durante 30 min. </li><li> Adicionar 10 ul de Tampão Proteinase K 10x (100 mM Tris-Cl pH 8,0, 50 mM de EDTA, pH 8,0, SDS a 5%). Misturar bem e adicionar 1 ml de Proteinase K. Incubar a 42 ° C durante 1 h. </li><li> Purificar DNA de entrada e chip usando kit comercial de purificação de PCR de acordo com o protocolo do fabricante e eluir DNA em 50 ul de água. ADN Armazenar a -20 ° C até à sua utilização. </li></ol> 2. Slot de Blot Análise de DNA ChIP <ol><li> Medir o rendimento de ADN de entrada e ChIP eluída em 50 ul de água, tal como descrito na etapa 1,22 utilizando um espectrofotómetro a 280 nm de acordo com o protocolo do fabricante. </li><li> Aqui volumes iguais (50 ul) de entrada ou de ADN imunoprecipitado que estão dentro do intervalo linear de detecção. <ol><li> Por exemplo, se a entradaRendimento de DNA é de 1,5 ng / mL, em seguida, tomar 30 ul (ou 45 ng de DNA total) e fazer volume para 50 mL com água. Para o fator Chip, tomar todo o fluido 50 ul do Passo 2.1 e vá para a etapa 2.3. </li></ol></li><li> Desnaturar 50 ul de entrada ou de ADN imunoprecipitado pela adição de 2,5 volumes de NaOH 0,4 N, vortex brevemente e centrifuga-se durante 956 xg durante 10 seg, e incubar à TA durante 30 min. </li><li> Neutraliza-se a ADN desnaturado através da adição de 175 ul de 1 M de Tris-HCl, pH 6,8, vortex, centrifugar para 956 xg durante 10 seg e colocar as amostras em gelo. </li><li> Prepara-se uma diluição em série de entrada e de ADN imunoprecipitado para o ensaio de transferência de entalhe, conforme descrito abaixo. <ol><li> Siga passos 2.3 - 2.4 para obter um volume total de amostra de 350 uL (ou seja, 50 ul de entrada / ADN Chip, 125 uL de NaOH 0,4 N, 175 ul de 1 M de Tris-HCl, pH 6,8). Nota: Para o exemplo acima referido, 45 ng de DNA de entrada vai acabar sendo 0,129 ng / mL. </li><li> Para DNA de entrada, rotular três tubes como 100, 50 e 25 e adiciona-se 100 ul, 50 ul e 25 ul da amostra de DNA desnaturada e neutralizada. Completar o volume final de 100 ul com água isenta de nuclease. Para chip de ADN, a etiqueta como três tubos 200, 100 e 50 e adicionar 200 ul, 100 ul e 50 ul da amostra de DNA desnaturada e neutralizada. Faça volume final para 200 mL com água livre de nuclease. </li></ol></li><li> Cortar a membrana com o tamanho do aparelho de slot blot. Pré-molhou a membrana e papéis mata-borrão em 20x SSC antes e antes da adição do DNA. Montá-los no aparelho de slot blot de acordo com o protocolo do fabricante. </li><li> DNA Pipet do passo 2.5.2 em slots apropriados. Aplicar vácuo para puxar lentamente o ADN sobre a membrana Zeta-probe. Pare de vácuo depois de todas as amostras passaram pelo aparelho. <ol><li> Desmontar o aparelho e imobilizar imediatamente o ADN para a membrana utilizando um reticulador de UV. Ter a cara da DNA-se no reticulador UV e usar o autocrosslink configuração em que o agente reticulante. Siga o protocolo do fabricante. </li></ol></li><li> Detectar BrdU no blot ranhura através da realização de transferência de western. <ol><li> Bloquear a membrana com 5% de leite seco não gordo em PBS contendo 0,1% de Tween-20 (PBST) durante 1 h à TA, para impedir a ligação não específica de anticorpos contra a membrana. </li><li> Adicionar anticorpo primário anti-BrdU (diluição 1: 500) diluída em 1% de leite seco não gordo em PBST e incubar o blot com o anticorpo primário durante 3 horas à temperatura ambiente num agitador. Lava-se a membrana com PBST três vezes após a incubação do anticorpo primário. </li><li> Adicionar anticorpo conjugado com HRP anti-ratinho secundário para o blot (1: 5000 de diluição) e incubar a temperatura ambiente durante 1 h. Lava-se a membrana com PBST três vezes após a incubação do anticorpo secundário. NOTA: Use um volume suficiente de anticorpos para cobrir completamente a membrana durante incubações de anticorpos primários e secundários. </li></ol></li><li> Preparar a mistura de ECL e adicioná-lo à ac blotgundo o protocolo do fabricante. Incubar a membrana com uma mistura de ECL durante 5 min à TA. <ol><li> Colocar a membrana em uma cassete de raios-X, expor a membrana a um filme de raios-X e desenvolver o blot utilizando o protocolo do fabricante. </li><li> Quantificar o sinal obtido para entrada e DNA chiped após a digitalização das manchas usando o software Image J de acordo com o protocolo do fabricante. Para quantificação, usar o sinal que está dentro da gama linear de detecção, a fim de garantir a precisão da medição. </li></ol></li></ol>

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I have no competing financial interests.

O protocolo descrito neste manuscrito é um método relativamente rápido para demonstrar a presença das proteínas ou as suas formas modificadas pós-tradução de DNA recentemente replicado ou nascente. Além disso, esta técnica permite um para medir a cinética da associação, dissociação de uma proteína ou a sua forma modificada com o ADN nascente. Esta técnica é complementar da tecnologia elegante iPOND 13. Na tecnologia iPOND, ADN sintetizado de novo é marcado com acetato de desoxiuridina (EdU). Um conjugado biotina é então adicionado para EdU usando o clique química. ADN nascente marcado com biotina é então imunoprecipitadas utilizando contas de estreptavidina e as proteínas co-purificação são detectados por Western blotting. Por outro lado, na nossa técnica de BrdU-Chip-Slot-ocidental, uma proteína ou a sua forma modificada associada com o ADN nascente é imunoprecipitada utilizando específico para a proteína ou o anticorpo específico da forma modificada e a quantidade de ADN nascente marcadas com BrdU ligado ao proteína é then determinada quantitativamente por transferência de Western utilizando-slot um anticorpo anti-BrdU. Há passos críticos neste protocolo que precisa de atenção especial. É crítico para assegurar que o anticorpo de interesse funciona bem em ensaios ChIP padrão com uma alta eficiência. É importante para testar a eficácia de um anticorpo em ensaios de chip ao quantitativo-PCR antes da realização do ensaio BrdU-Chip-entalhe Ocidental. Para a quantificação da técnica-BrdU-Chip-entalhe ocidental para ser preciso, uma diluição em série de ADN marcado com BrdU deve ser aplicado para blot ranhura e análise de Western utilizando o anticorpo anti-BrdU deve ser inicialmente realizado a fim de determinar a gama linear de detecção. Determinação da concentração de ADN de chip e ADN de entrada permite que se garantir que a quantidade de ADN marcado com BrdU está dentro do intervalo linear de detecção em Western blotting. Por exemplo, determinou-se 50 ng e 12,5 ng de ADN de entrada para aa gama linear em nossa experiência-piloto. Além disso, se a concentração das amostras de aparas é muito elevado, é imperativo para diluir o ADN de chip antes da realização de ranhura. A limitação desta técnica é a sua resolução, a qual é dependente da eficiência de corte da cromatina. A tosquia da cromatina por sonicação determina a proximidade de uma dada proteína, ou a sua forma modificada para o ADN recém-sintetizado. No passado, o estudo das mudanças na cromatina e enzimas modificadoras da cromatina durante a replicação do DNA em células de mamíferos permaneceu uma pergunta difícil de abordar, devido à indisponibilidade de técnicas adequadas. Além disso, uma vez que as células Hdac1,2-nulos de captura em fase G1, que era difícil de examinar funções Hdac1,2 para dentro da fase S. Em nosso estudo, mostramos as funções para estes dois HDACs durante a replicação do DNA usando uma combinação de inibidores seletivos primeiro-in-class para inibir suas atividades dentro da fase S e do romance BrdU-chip-Stécnica muito ocidental. No futuro, esta técnica também pode ser usada para estudar a dinâmica das proteínas de resposta de dano de ADN e de reparação do ADN na bifurcação parado além de estudar as modificações de histonas e enzima de modificação de cromatina na bifurcação. Além disso, esta técnica não é limitada às células do rato NIH3T3. Esta técnica pode ser utilizada com sucesso em outras linhas de células, a fim de investigar como outros inibidores de enzimas ou proteínas de afectar a replicação de ADN e replicação alterações induzidas pelo stress cromatina no garfo de replicação.

Reparação de ADN defeituosos e / ou a replicação de DNA são uma das principais causas de instabilidade do genoma, o que pode provocar a morte celular. Uma única quebra de cadeia dupla unrepaired é suficiente para causar morte celular 1. Organização da cromatina é transitoriamente alterada durante a replicação e reparar 2,3, e falha em manter a informação epigenética durante esses processos irão resultar em uma ameaça à integridade do genoma. Perda de HDAC3 ou função HDAC1,2 impede a progressão da fase S, replicação e reparação do ADN, levando ao estresse genotóxico (dano ao DNA) e morte celular 4-9. É, por conseguinte, uma estratégia prático utilizar os inibidores de HDAC selectivos para interromper a replicação, reparação e cromatina em células cancerosas e causar danos no ADN, que por sua vez pode parar o crescimento e induzir a morte celular selectiva, em que crescem rapidamente células cancerosas. Durante a replicação do DNA, cromatina é rapidamente desmontada e remontada em seguida, seguir a duplicação do DNA. Recém-synthesized histona H4 acetilada é a K5 e K12 (H4K5K12ac resíduos) e a seguir para a deposição de cromatina, são rapidamente desacetilado por histona desacetilases (HDACs) 10,11. A falha das células para manter a integridade da cromatina nascente pela histona desacetilação pode conduzir ao colapso da forquilha, que por sua vez pode resultar em danos no ADN e morte celular. Nós recentemente mostrou que a inibição seletiva de HDAC1,2 aumenta acetilação das histonas (H4K5ac, H4K12ac e H4K16ac) e inibe a atividade SMARCA5 cromatina remodeler na cromatina nascente, que se correlaciona com a redução da progressão da forquilha de replicação, maior colapso garfo e aumento da replicação do estresse induzido por danos no DNA 6 . Assim, o tratamento com inibidor de HDAC podem alterar a estrutura da cromatina nascente para provocar danos no ADN e morte muito rapidamente em células de cancro, uma vez que estas células ciclo rapidamente e muitas vezes passar através da fase-S. Por isso, é importante entender como HDACs funcionar para regular acetilação das histonas e bindi proteínang de manter replicação de ADN em células de mamífero. Para medir quantitativamente a quantidade de acetilação das histonas e SMARCA5 remodeler cromatina associada à DNA durante a replicação do DNA nascente, eu inventei um ensaio ChIP modificada chamada a técnica de BrdU-chip-Slot-ocidental. Após imunoprecipitação da cromatina (ChIP) de uma modificação da proteína ou histona desejado, a quantidade de ADN nascente (marcado utilizando análogo da timidina, BrdU) na amostra de chip pode ser detectada por meio de análise ocidental da marcadas com BrdU chip de ADN transferido para uma membrana utilizando um entalhe Aparelho Blot. Usando esta técnica, nós mostramos que H4K16ac (a marca envolvido em embalagem cromatina) eo membro da família ISWI SMARCA5 (dependente de actina regulador associada à matriz relacionada com o SNF SWI / da cromatina) remodeler cromatina estão associados com DNA nascente em células em fase S 6. Também descobrimos que a marca H4K16ac nascente é desacetilada por HDAC1,2 durante a replicação do DNA 6. Inibe H4K16acatividade remodeler cromatina do membro da família ISWI SMARCA5 12. Assim, usando a técnica de-BrdU-CHIP-Slot Ocidental, podemos conectar as funções para HDAC1,2 na regulação da remodelação da cromatina durante a replicação do DNA. Assim, a técnica BrdU-chip-Slot-Western Blot é uma abordagem poderosa para medir quantitativamente a associação e dissociação dinâmica de proteínas ou suas modificações pós-traducionais que são obrigados a DNA nascente.

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-BrdU (westerns)</td>
			<td style="width:107px;">BD Biosciences</td>
			<td style="width:119px;">B555627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SMARCA5</td>
			<td>Abcam</td>
			<td style="width:119px;">ab3749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-H4K16ac</td>
			<td style="width:107px;">Active Motif</td>
			<td>39167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeta-Probe GT Membrane</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">162-0197</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formaldehyde</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:119px;">BP531-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protein A agarose</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;">16-156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase A</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:119px;">19101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">P4850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PCR Purification Kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:119px;">28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">G7403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:107px;">Roche</td>
			<td style="width:119px;">11836170001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BrdU</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">B9285</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit IgG&#160;</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;">12-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">S-3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton-X-100</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">93443</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NP40</td>
			<td style="width:107px;">USB Corporation</td>
			<td style="width:119px;">19628</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium deoxycholate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">D6750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">S-4019</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol</td>
			<td style="width:107px;">Decon Laboratories</td>
			<td style="width:119px;">04-355-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DEPC-treated water</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">95284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:119px;">BP-152-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:107px;">Ambion</td>
			<td style="width:119px;">AM9820</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium hydroxide</td>
			<td style="width:107px;">Mallinckrodt GenAR&#160;</td>
			<td style="width:119px;">MAL7772-06</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20X SSC</td>
			<td style="width:107px;">Life Technologies</td>
			<td style="width:119px;">15557-036</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Non-fat dry milk</td>
			<td style="width:107px;">Lab Scientific Inc</td>
			<td style="width:119px;">M0841</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ECL</td>
			<td style="width:107px;">Thermo Fisher</td>
			<td style="width:119px;">80196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">X-ray film</td>
			<td style="width:107px;">Genesee Sci</td>
			<td style="width:119px;">30-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Developer</td>
			<td style="width:107px;">Konica Minolta</td>
			<td style="width:119px;">SRX-101A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV Crosslinker</td>
			<td style="width:107px;">Stratagene</td>
			<td style="width:119px;">XLE-1000</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Sonicator</td>
			<td style="width:107px;">Branson Sonifier&#160; Digital Ultrasonic Cell Disruptor</td>
			<td style="width:119px;">Model: 450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:119px;">Model: 5810R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Water bath</td>
			<td style="width:107px;">Fisher Scientific Isotemp</td>
			<td style="width:119px;">Model: 2322</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoDrop 1000 spectrophotometer</td>
			<td style="width:107px;">ThermoScientific</td>
			<td style="width:119px;">Model: 1000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Slot Blot apparatus&#160;</td>
			<td style="width:107px;">Schleicher and Schuell Minifold II</td>
			<td style="width:119px;">44-27570</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Tissue Culture Incubator</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:119px;">Series II 3110 Water-Jacketed CO2 Incubators</td>
			<td></td>
		</tr>
	</tbody>
</table>

examination of proteins bound to nascent dna in mammalian cells using  brdu-chip-slot-western technique

Histona-desacetilases 1 e 2 (HDAC1,2) localizam os sítios de replicação do ADN. No estudo anterior, utilizando-se um inibidor selectivo e um sistema genético knockdown, que mostrou novos funções para HDAC1,2 na replicação garfo progressão e manutenção nascente cromatina em células de mamífero. Além disso, usamos uma técnica BrdU-chip-Slot-ocidental que combina imunoprecipitação da cromatina (ChIP) de bromo-desoxiuridina (BrdU) DNA marcado com com slot blot e análise Ocidental para medir quantitativamente proteínas ou modificação da histona associadas com DNA nascente. Activamente células em divisão foram tratados com HDAC1,2 inibidor selectivo ou transfectadas com ARNsi contra HDAC1 e HDAC2 e, em seguida, o ADN recém-sintetizado foi marcado com o análogo de timidina bromodesoxiuridina (BrdU). A marcação com BrdU foi realizada em um ponto de tempo quando não havia paragem do ciclo celular ou apoptose significativa devido à perda de funções HDAC1,2. Seguindo lAbeling de células com BrdU, a imunoprecipitação da cromatina (ChIP) de marcas de acetilação de histonas ou da cromatina-remodeler foi realizada com anticorpos específicos. Entrada de ADN marcadas com BrdU e o imunoprecipitado (ou chiped) DNA foi então manchado para uma membrana utilizando a técnica de fenda blot e imobilizada utilizando UV. A quantidade de ADN nascente em cada slot foi depois avaliado quantitativamente usando análise de Western com um anticorpo anti-BrdU. O efeito da perda de funções HDAC1,2 sobre os níveis de histonas recentemente sintetizadas marcas de acetilação associado com ADN e cromatina remodeler foi então determinada através da normalização do sinal de BrdU-chip obtidos a partir das amostras tratadas para as amostras de controlo.

Para determinar a especificidade dos inibidores selectivos HDAC1,2, foram usadas Hdac1,2 FL / FL e FL HDAC3 / FL células de fibrossarcoma. Foi utilizado a recombinase Cre (Ad-Cre) contendo adenovírus para excluir Hdac1,2 e HDAC3 nestas células. Após a infecção Ad-Cre de Hdac1,2 FL / FL células, foi observado um aumento robusto na H4K5ac. O tratamento de células com knockout Hdac1,2 233 ou 898, não resultou em qualquer aumento adicional na H4K5ac confirmando que 233 e 898 e não inibem Hdac1,2 HDAC3 nestas células. Em contraste, a adição de 233 ou 898 para as células knockout HDAC3 resultou num aumento significativo na H4K5ac em comparação com o aumento observado nas células knockout HDAC3 devido a um efeito aditivo sobre os níveis H4K5ac como um resultado da inibição de Hdac1,2 e 3. Assim, 233 e 898 são HDAC1, inibidores da 2-selectivos. Estes resultados são mostrados nasFigura 1. Para validar a técnica BrdU-chip-Slot, análise BrdU-pulse chase foi realizada a olhar para a cinética de PCNA carregamento para o DNA nascente em células HeLa. Nossos resultados mostraram que a associação com PCNA DNA nascente ocorre rapidamente dentro de 15 minutos e desaparece depois de 30 minutos de perseguição, de acordo com resultados publicados anteriormente 13. Estes resultados são mostrados na Figura 2. Técnica BrdU-H4K16ac ChIP-Slot-de Western foi utilizada para determinar a quantidade de H4K16ac associada com o ADN nascente, na ausência da função HDAC1,2. Um enriquecimento robusta em H4K16ac associada com o ADN nascente foi observada quando em comparação com o controlo de IgG de coelho (Figuras 3A e 3B). O aumento na nascente H4K16ac-ADN associado com a inibição das actividades HDAC1,2 ou knockdown de Hdac1,2 é mostrado na Figura 3C </strong&gt;, 3D e 3E. O nível de SMARCA5 cromatina remodeler no DNA nascente foi determinada utilizando-BrdU SMARCA5 técnica de ChIP-Slot Ocidental. Os nossos resultados mostraram que os associados com o ADN nascente SMARCA5 em células de mamífero. Além disso, a inibição ou eliminação de HDAC1,2 Hdac1,2 não alterou a quantidade de ADN nascente associados-SMARCA5 cromatina remodeler como mostrado na Figura 4. <img alt="figura 1" src="/files/ftp_upload/53647/53647fig1.jpg" /> Figura 1. Confirmação da especificidade dos inibidores selectivos HDAC1,2. Análise de Western de lisados ​​de células totais preparados a partir de HDAC1 FL / FL HDAC2 FL / FL ou células de fibrossarcoma HDAC3 FL / FL seguintes infecção Ad-Cre e 898 ou com o tratamento233. As células foram tratadas com 3? M 898 ou 233, durante 24 h, 48 h após uma infecção Ad-Cre. Este valor é derivado do nosso anterior trabalho publicado 6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53647/53647fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53647/53647fig2.jpg" /> Figura 2. Dinâmica do PCNA associação com o DNA Nascente em células HeLa. Células HeLa foram marcadas com bromodeoxiuridina (BrdU) durante 30 min. As células foram então lavadas para remover não incorporada BrdU e cultivadas em meio sem BrdU por períodos de tempo indicados (Chase). Imunoprecipitação da cromatina (ChIP) foi realizada com o antígeno nuclear de célula anti-proliferante (PCNA) anticorpo. ADN marcado com BrdU presente no DNA de entrada e as associadas com o PCNA foram avaliados na análise de mancha de slot utilizando um anti-BRDU anticorpo como mostrado na Figura 2. Este valor é derivado do nosso anterior trabalho publicado 6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53647/53647fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53647/53647fig3.jpg" /> Figura 3. Perda da histona-desacetilase 1 e 2 aumenta H4K16ac em ADN nascente. (AB) Bromodeoxiuridina (BrdU) perseguição de impulso foi realizado em células HeLa e NIH3T3 para determinar associação de H4K16ac com o ADN nascente nos pontos de tempo indicados. (CD) células NIH3T3 foram tratadas com DMSO ou um inibidor selectivo HDAC1,2 (898 ou 233), durante 24 h. Células (E) NIH3T3 ou foram transfectadas com não-alvo (NT) ou Hdac1,2 (H12) siRNA. As células a partir de (C - E) foram marcadas com BrdU e utilizadospara chip com anti-H4K16ac seguido por slot blot. A membrana foi sondada com anticorpo anti-BrdU. Os números indicam a Imagem J quantificação do sinal média BrdU do volume alto e médio de DNA ChIP manchado. Este valor é derivado do nosso anterior trabalho publicado 6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53647/53647fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53647/53647fig4.jpg" /> Figura 4. SMARCA5 Associates com DNA Nascente. A quantidade de SMARCA5 na cromatina nascente seguinte perda de função HDAC1,2 é mostrado. Células NIH3T3 ou foram tratados com um inibidor selectivo HDAC1,2 (898) ou transfectadas com não-alvo (NT) ou Hdac1,2 (H12) siRNA. As células foram marcadas com BrdU após os tratamentos e chip acima mencionados com anti-IgG de coelho SMARCA5 ou (c negativoontrolo) foi realizada. Volumes crescentes de ADN de chip foi manchada sobre o blot ranhura e a membrana foi sondada com anticorpo anti-BrdU. Este valor é derivado do nosso anterior trabalho publicado 6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53647/53647fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique at JoVE.com

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Exame de proteínas ligadas ao DNA em células de mamíferos Nascente usar a técnica de-BrdU-chip-Slot Ocidental

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

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9.1K visualizações.  University of Utah School of Medicine. In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA. 

Vídeo: Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Longo prazo células de mamíferos utilizando imagens do Largo-Campo Microscopia

Biologia

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Transfecção de DNA de mamíferos usando músculos esqueléticos In Vivo Eletroporação

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Intranucleares microinjeção de DNA em Dissociated neurônios de mamíferos adultos

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

Análise de DNA dupla fita-Break Repair (DSB) em células de mamíferos

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Visualização de replicação de DNA no DT40 Vertebrados Modelo de Sistema utilizando a técnica de DNA Fibra

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usando Seqüência de Detenção SECM como uma ferramenta para isolar Ribossomos Polipeptídeos encadernados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Identificação de parceiros de interação de proteínas em células de mamíferos utilizando SILAC-imunoprecipitação quantitativos Proteômica

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Uma interface de usuário gráfica para rastreamento assistido por software da concentração de proteínas em protrusões celulares dinâmicas

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...