Este trabajo fue apoyado por el NIH subvenciones CA141009 y GM39067. Agradecemos a E. Parnell y R. Yarrington los comentarios sobre la parte escrita.

1. Aislamiento de ratón esplénica ingenuo CD4 de células T <ol><li> Sacrificar el ratón de una manera humana en consonancia con los protocolos de uso del Comité (IACUC) Institucional Cuidado de Animales y. Disección del bazo y colóquelo en un plato de Petri que contiene 10 ml de DMEM con 10% de SFB. </li><li> Machacar el bazo usando extremos helados de dos placas de vidrio para liberar los esplenocitos. Transferir la suspensión celular en un tubo cónico de 15 ml. </li><li> Recoger las células por centrifugación a 200 xg (~ 1200 rpm para una centrífuga clínica con un diámetro típico del rotor) durante 5 min a 4 ° C. </li><li> Resuspender las células en 2 ml de tampón ACK para lisar las células rojas de la sangre, 1 min a temperatura ambiente (RT). Detener la reacción mediante la adición de 8 ml de DMEM con FBS al. </li><li> Recoger las células por centrifugación a 200 xg durante 5 min a 4 ° C. </li><li> Resuspender las células en 5 ml de DMEM que contenía FBS y pasar a través de un filtro de malla de 70 micras (BD Falcon, Cat # 352350). Contar las células y procederpara el aislamiento de células T CD4 + naïve utilizando un kit de aislamiento de CD4 (Miltenyi, Cat # 130-095-248) utilizando las instrucciones del fabricante. Recoger ingenuos células T CD4 por centrifugación a 200 xg durante 5 min a 4 ° C. Resuspender las células en 10 ml de DMEM con FBS al. </li></ol> 2. Preparación de la cromatina <ol><li> Añadir 37% de formaldehído a una concentración final de 1% a la suspensión celular en DMEM y agite con cuidado a temperatura ambiente (por ejemplo, utilizando un Nutator) durante 15 min para reticular complejos ADN: proteína. </li><li> Deja de reticulación mediante la adición de 1 M de glicina a una concentración final de 125 mM. Continuar a la roca durante 5 minutos a temperatura ambiente. </li><li> Recoger las células por centrifugación a 200 xg durante 5 min a 4 ° C. </li><li> Lavar las células mediante resuspensión en 5 ml de inhibidores de proteasas que contienen PBS enfriado con hielo. Lavar con inhibidores de la proteasa que contienen helado de PBS 3 veces en total y recoger las células por centrifugación a 4 ° C. </li><li> Resuspender las células en 1 mltampón de lisis celular de hielo frío que contiene inhibidores de la proteasa. Incubar en hielo durante 15 min. La eficiencia de la lisis de las células se puede determinar mediante la resuspensión de una pequeña alícuota de las células en solución de azul de tripano al 0,4% (Sigma, Cat # T8154) y la observación con un microscopio. </li><li> Recoger los núcleos por centrifugación a 200 xg (normalmente ~ 1200 rpm) durante 5 min a 4 ° C. Elimine con cuidado el sobrenadante. </li><li> Resuspender los núcleos en 500 l de tampón de lisis nuclear que contiene inhibidores de la proteasa. Incubar en hielo durante 15 min. </li><li> Sonicar las células por medio de un aparato de ultrasonidos Misonix 3000, tamaño de la sonda 1,6 mm: Nivel de salida 4, 15 seg ráfaga, 4 veces en el hielo. Cada muestra debe ser enfriado en hielo durante 1-2 minutos antes de la sonicación de nuevo para evitar el sobrecalentamiento de las muestras. El recalentamiento puede causar la reversión de enlaces cruzados. </li><li> Centrifugar la cromatina sometida a ultrasonidos a 16.000 xg (~ 13.200 rpm en una microcentrífuga) durante 5 min a 4 ° C. </li><li> Tomar una alícuota de 20 l supern claraatant, añadir colorante de carga de ADN y comprobar el ADN sonicado por electroforesis a través de un gel de agarosa al 2% (tamaño ideal de ADN sonicado para la mayoría de aplicaciones es de 200-500 pb). </li><li> Determinar la concentración de ADN utilizando un espectrofotómetro UV. Cromatina esquilada se puede utilizar inmediatamente para establecer la reacción de inmunoprecipitación de cromatina y se almacenan a -80 ° C. Normalmente se obtiene 7,5-10 g de ADN 2-3 x 10 6 células T CD4 purificadas por ratón. </li></ol> 3. Inmunoprecipitación de cromatina (Todos los pasos deben llevarse a cabo a 0-4 º C) <ol><li> Diluir sonicated cromatina a una concentración de ADN de mg / ml 5-10 (volumen total = 1 ml) en tampón de dilución chip con los inhibidores de la proteasa. </li><li> Guardar 100 l (10%) como entrada. Almacenar en hielo. </li><li> Alícuota de 450 l cada uno en dos tubos de microcentrífuga de 1,7 ml etiquetados como control de isotipo (IgG de conejo o ratón) y anticuerpo de interés. Si se utilizan múltiples anticuerpos del mismo isotipo, una sola contr isotipool será suficiente para realizar este análisis. Usted tendrá múltiples controles de isotipo si se utilizan anticuerpos de una fuente animal diferente o isotipo. </li><li> Añadir 2-5 g de anticuerpo específico, dependiendo de la especificidad del anticuerpo usado, o control de isotipo a los tubos respectivos. </li><li> Roca los tubos utilizando un Nutator durante la noche a 4 ° C para permitir la formación de complejos de la cromatina-anticuerpo. </li><li> Añadir 25 l de perlas de proteína G magnéticos (01:01 emulsión de gránulos en suspensión) a la mezcla anterior y permitir a la roca durante al menos 2 horas a 4 ° C. Cuentas de nuestro proveedor se puede utilizar directamente. </li><li> Coloque los tubos de microcentrífuga en un soporte magnético y permiten que las perlas se recogen en la parte magnetizada. </li><li> Retire con cuidado la solución por aspiración sin molestar a los granos. </li><li> Añadir 1 ml de solución de lavado bajas sal y deje mecer suavemente durante 5 min en un Nutator. Recoger las perlas usando un soporte magnético y eliminar la solución de lavado. Repetir una vez.</li><li> Añadir 1 ml de solución de lavado de sal y se deja mecer por 5 min en una Nutator. Recoger las perlas usando el soporte magnético y eliminar la solución de lavado. Repetir una vez. </li><li> Añadir 1 ml de solución de lavado de cloruro de litio y permitir a la roca durante 5 minutos en una Nutator. Recoger las perlas usando el soporte magnético y eliminar la solución de lavado. Repetir una vez. El uso de LiCl mejora la eliminación eficaz de las interacciones no específicas de cromatina con las perlas. </li><li> Añadir 1 ml de solución de TE y se deja mecer por 5 min en una Nutator. Recoger las perlas usando el soporte magnético y eliminar la solución de lavado. </li><li> Eluir el ADN a partir de las perlas mediante la adición de 250 l de tampón de elución. Roca durante 15 minutos a temperatura ambiente. Pipetear fuera el líquido eluido y guardar este material en un nuevo tubo de microcentrífuga de 1,7 ml. Repetir una vez más y se combinan tanto las elusiones. Deseche las cuentas. </li><li> Para invertir las reticulaciones añaden 5 M de NaCl a una concentración final de 0,3 M y 1 l de RNasa A (20 m<html> g / ml) al ADN eluido. Añadir 400 l de tampón de elución a las entradas guardadas en la etapa 3.2, para hacer que el volumen de 500 l. Para las entradas de añadir NaCl hasta 0,3 M, 1 l de RNasa A, 10 l de EDTA 0,5 M, 20 l de 1 M Tris-HCl, pH 6,5 y 1 l de proteinasa K (20 mg / ml). </li><li> Incubar los tubos durante la noche a 65 ° C en un bloque de calentamiento en seco. Para evitar la evaporación de las muestras, sello o colocar un peso en los tubos para evitar que se abra. Un horno de hibridación también se puede utilizar. </li><li> Deje enfriar los tubos a temperatura ambiente. Añadir 1 ml de etanol al 100% y se incuba 2 horas-durante la noche a -80 ° C para precipitar el ADN. </li><li> Centrifugar los tubos a 16.000 xg durante 15 min para sedimentar el ADN. Lavar el sedimento de ADN una vez con etanol al 70% y secado al aire el sedimento. Resuspender el sedimento de ADN en 100 l de agua destilada tratada en autoclave. </li><li> Purificar ADN utilizando columnas QIAquick Spin y eluyen en el volumen total de 50 l de tampón de elución. Este ADN está listo para su uso para la PCR. </li></ol>&quot;&gt; 4. Preparación de la reacción de PCR (Todos los pasos deben llevarse a cabo en el hielo) <ol><li> Recoge todas las muestras de ADN de muestras de entrada y chip correspondiente. También, recoger todos los reactivos y cebadores para la región objeto de PCR, así como una región de control. Use un &quot;hotstart&quot; Taq ADN polimerasa. Vamos a utilizar un ensayo basado en SYBR Green-para cuantificar la amplificación de ADN en este procedimiento, pero los kits qPCR Mastermix se puede utilizar si es necesario. </li><li> Hacer una dilución en serie de ADN de entrada para generar una curva estándar en el análisis de qPCR: por ejemplo, 10%, 1%, 0,1% y 0,01% en tampón de elución (de las columnas QIAquick Spin en el paso 3,18). El grado de concentración y aumento de la plantilla conjunto curva estándar pueden variar en función de los niveles esperados de enriquecimiento chip, etc dispensación 10 l de muestras de ADN de entrada en los respectivos pocillos de una placa de 96 pocillos (Genemate, cat # T-3182-1 ), por duplicado. </li><li> Dispensar 10 ml de ADN de chip de control de isotipo, así como anticuerpos específicos en la respectivapozos, por triplicado. </li><li> Hacer una &quot;mezcla maestra&quot; que contiene cualquiera de los cebadores o cebadores de control de destino (0,1 mM concentración final de cada cebador) y prescindir de 10 l en pocillos respectivos. Por ejemplo, en la plantilla a continuación dispensar maestro de la mezcla que contiene cebadores específicos en los pocillos marcado en verde y dispensar la mezcla principal con cebadores de control en los pocillos marcados en rojo. </li><li> Cubrir la placa de PCR con película de sellado plástico óptico y centrifugar a 500 xg en una centrífuga clínica (~ 1200 rpm) durante 1 min a temperatura ambiente para recoger todo lo que en la parte inferior del pozo. </li><li> Inicie la reacción de PCR. Vamos a utilizar el LightCycler 480 II (Roche) que operan LightCycler 480SW 1.5 software para ejecutar la qPCR en este ejemplo. </li></ol> Pre-incubación: 1 ciclo de: 95 º C / 5 min. Amplificación: 40 a 45 ciclos de: 94 º C / 5 seg, 60 ° C / 5 seg, 72 ° C/10 seg (temperatura de hibridación debe ser 2-3 º C inferior a la temperatur fusióne de los cebadores). Curva de fusión: 1 ciclo. Enfriamiento: 1 ciclo. <table border="1"><tbody><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 11 </td><td> 12 </td></tr><tr><td> La </td><td> 10% de entrada </td><td> 10% de entrada </td><td> Isotipo </td><td> Específico </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> B </td><td> 1% de entrada </td><td> 1% de entrada </td><td> Isotipo </td><td> Específico </td><td></td><td></td><td></td><td></td><td></td><td></td&gt; <td></td><td></td></tr><tr><td> C </td><td> 0,1% de entrada </td><td> 0,1% de entrada </td><td> Isotipo </td><td> Específico </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> D </td><td> 0,01% de entrada </td><td> 0,01% de entrada </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> E </td><td> 10% de entrada </td><td> 10% de entrada </td><td> Isotipo </td><td> Específico </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> F </td><td> 1% de entrada </td><td> 1% de entrada </td><td> Isotipo </td><td> Específico </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> T </td><td> 0,1% de entrada </td><td> 0,1% de entrada </td><td> Isotipo </td><td> Específico </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> H </td><td> 0,01% de entrada </td><td> 0,01% de entrada </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> Verde: Use cebadores de la región objetivo. Rojo: Use cebadores de la región de control. 5. Análisis <ol><li> Programa de software de la qPCR para la cuantificación de la cantidad absoluta de ADN. Es importante destacar que, el uso de una curva estándar para interpolar las cantidades de ADN en las muestras específicas y isotipo desconocidos permite la evaluación y la congruencia de la calidad y la eficiencia de todo el cebador se pone sobre la gama de concentraciones que se encuentran en las muestras. Por otra parte, también proporciona una mortalidade método fiable para convertir los valores Cq (C t o C p) a los resultados de enriquecimiento veces en relación finales que los métodos usingΔΔCq y afines. </li><li> Ir al editor de la muestra y marcar los pocillos que contienen muestras de entrada de varias diluciones (10%, 1%, 0,1% y 0,01%) con sus valores de la curva estándar. También etiquetar los pocillos con muestras de chip desconocidos, tal y como la placa de PCR se presenta. En el software equivalente utilizado en otras máquinas qPCR, designar subconjuntos correspondientes a las reacciones de cada par de cebadores, valor de la curva estándar y la muestra experimental y control de isotipo correspondiente. </li><li> En el software LightCycler, ir al editor de subconjunto y marcar los subconjuntos incluyendo los pocillos con muestras de entrada, así como las muestras de chip desconocidos. Las muestras desconocidas se cuantificaron utilizando la curva estándar generada a partir de las concentraciones conocidas de muestras de entrada para el análisis. En el software equivalente utilizado en otras máquinas qPCR, designado subconjuntos corresponding a todas las reacciones para cada uno de los pares de cebadores. </li><li> Después de la amplificación por PCR es completa, realizar el análisis con &quot;Abs Quant/2nd Derivado Max&quot; y seleccionar el subconjunto de análisis y pulse Aceptar. En el software equivalente utilizado en otras máquinas qPCR, convertir el valor Cq a la cantidad de ADN en cada subgrupo. </li><li> Pulse &quot;Calcular&quot;, que va a generar la curva estándar utilizando concentraciones conocidas de las muestras de entrada y mostrará la cantidad absoluta de ADN presente en las muestras desconocidas. </li><li> Si la calidad del ADN cizallado es buena, y si los cebadores se unen específicamente a la región objetivo, la eficiencia de PCR debe estar cerca de 2. </li><li> Realizar un análisis de la curva de fusión con &quot;Tm Calling&quot; para el mismo subconjunto, si está disponible. Un único pico indica que sólo un producto específico se amplificó. La amplificación de ADN específico también se puede probar mediante electroforesis del producto de PCR junto con escalera de ADN a través de un gel de agarosa. </li><li> Exportar datos como por tabuladoresarchivo de texto delimitado, que se puede abrir en Microsoft Excel para su posterior análisis. </li><li> Utilizando Microsoft Excel, divida la cantidad de ADN para el anticuerpo específico con el control de isotipo de primers específicos. Repita este paso para los cebadores de la región de control. Si se utilizan múltiples anticuerpos del mismo isotipo para diferentes inmunoprecipitaciones, normalizar cada uno de estos con los valores de la misma control de isotipo. Por otra parte, si se utilizan múltiples pares de cebadores específicos, las cantidades de ADN de un único par de cebadores de control de conjunto se deben utilizar para la normalización de cada objetivo (Figuras 1 y 2). Los valores de salida representan el anticuerpo inmunoprecipitación veces enriquecimiento específico en cada ubicación con respecto a los anticuerpos no específicos fondo inmunoprecipitación. </li><li> Divida el enriquecimiento veces (al isotipo control) de primers específicos con el enriquecimiento veces (al isotipo control) de cebadores de la región de control para obtener el enriquecimiento relativo a unregión no unida de control (Figura 1). Es importante destacar que, en cuenta que debido a la generación de las curvas de calibración con el total de ADN de entrada para cada muestra, cada uno de los valores interpolados a partir de inmunoprecipitación por encima de las curvas estándar ya están normalizados a, y se expresaron como, la fracción del total de entrada por el software de la máquina. </li><li> Si el rigor de los tampones utilizados para la inmunoprecipitación o lavado es demasiado alta, es posible que se observó amplificación robusta a partir de muestras inmunoprecipitadas con anticuerpos específicos, mientras que la amplificación del control de isotipo inmunoprecipitó no se observaron muestras. En tal escenario, es mejor que restar la cantidad de chip de control de isotipo de chip de anticuerpos específicos tanto para la región específica y la región no unida. Enriquecimiento relativo puede calcularse dividiendo la diferencia de la región de orientación por diferencia de región no unida (Figura 3). </li></ol>

<ol>
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No hay conflictos de interés declarado.

El protocolo anterior proporciona un método robusto de cuantificar con precisión enriquecimiento de ADN a partir de linfocitos primarios que utilizan chip. Una de las principales razones de robustez en este protocolo es la inclusión de repeticiones biológica. El protocolo anterior utiliza tres repeticiones, el enriquecimiento de la cual se calcula de forma independiente. Las salidas se promedian para proporcionar un grado de enriquecimiento y las desviaciones estándar calculadas para proporcionar una medida de la v...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>Sigma</td>
 <td>F-8775</td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline</td>
 <td>Hyclone</td>
 <td>SH30256.01</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Protease Inhibitor tablets</td>
 <td>Roche</td>
 <td>04693116001</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Protein G magnetic beads</td>
 <td>Active Motif</td>
 <td>101945</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>RNase A (20 mg/ml)</td>
 <td>EMD Millipore</td>
 <td>556746</td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Proteinase K (20 mg/ml)</td>
 <td>Roche</td>
 <td>03115879001</td>
 <td>Dissolve in 50 mM Tris-HCl, 10 mM CaCl2, pH 8.0</td>
 </tr>
 <tr>
 <td>Platinum Taq DNA Polymerase</td>
 <td>Invitrogen</td>
 <td>10966-034</td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>SYBR Green I</td>
 <td>Invitrogen</td>
 <td>S7567</td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>1 M Glycine</td>
 <td></td>
 <td></td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td>Cell lysis buffer (5 mM Pipes, pH 8.0; 85 mM KCl; 0.5% NP-40)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Nuclear lysis buffer (50 mM Tris, pH 8.1; 10 mM EDTA; 1% SDS)</td>
 <td></td>
 <td></td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td>ChIP dilution buffer (0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris pH 8.1; 190 mM NaCl)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Low salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris pH 8.1; 150 mM NaCl)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>High salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris pH 8.1; 600 mM NaCl)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>LiCl wash buffer (0.25 M LiCl; 1% NP-40; 1% Sodium Deoxycholate, 1 mM EDTA; 10 mM Tris pH 8.0)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Elution buffer (1% SDS; 0.1 M NaHCO3)</td>
 <td></td>
 <td></td>
 <td>Prepare fresh</td>
 </tr>
 <tr>
 <td>5 M NaCl</td>
 <td></td>
 <td></td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td>0.5 M EDTA</td>
 <td></td>
 <td></td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td>1 M Tris-HCl, pH 6.5</td>
 <td></td>
 <td></td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table of Specific Reagents</td>
 </tr>
 <tr>
 <td>Clay Adams Brand Nutator</td>
 <td>Becton Dickinson</td>
 <td>Model: 421105</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnetic Stand</td>
 <td>Promega</td>
 <td>Z5342</td>
 <td></td>
 </tr>
 <tr>
 <td>Qiaquick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28106</td>
 <td></td>
 </tr>
 <tr>
 <td>Masonix Sonicator 3000</td>
 <td>QSonica</td>
 <td>Model: S3000</td>
 <td></td>
 </tr>
 <tr>
 <td>UV Spectrophotometer</td>
 <td>NanoDrop Technologies</td>
 <td>ND-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Heating Block</td>
 <td>VWR</td>
 <td>13259-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotator</td>
 <td>VWR</td>
 <td>80085-692 </td>
 <td></td>
 </tr>
 <tr>
 <td>Refrigerated bench top centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Model: Allegra X-12R</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td>Eppendorf</td>
 <td>5415 D</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table of Equipment </td>
 </tr>
 </tbody>
</table>

efficient chromatin immunoprecipitation using limiting amounts of biomass

Cromatina immunoprecipitation (CHIP) es un método ampliamente utilizado para la determinación de las interacciones de las diferentes proteínas con el ADN en la cromatina de las células vivas. Los ejemplos incluyen la secuencia específica de unión al ADN, histonas factores de transcripción y sus diferentes estados de modificación, enzimas tales como polimerasas de ARN y factores auxiliares, y los componentes de reparación del ADN. A pesar de su ubicuidad, hay una falta de up-to-date, metodologías detalladas, tanto para la preparación del banco de material y para un análisis preciso permitir mediciones cuantitativas de interacción. Debido a esta falta de información, y también porque, como cualquier inmunoprecipitación, las condiciones deben ser re-optimizado para los nuevos conjuntos de condiciones experimentales, el chip de ensayo es susceptible a resultados imprecisos o mal cuantitativa. Nuestro protocolo se deriva en última instancia del trabajo seminal en el factor de transcripción: interacciones ADN 1,2, pero incorpora una serie de mejoras a la sensividad y la reproducibilidad de difíciles a obtener tipos de células. El protocolo se ha utilizado con éxito 3,4, tanto utilizando qPCR para cuantificar enriquecimiento de ADN, o el uso de una variante semi-cuantitativa de la siguiente protocolo. Este análisis cuantitativo de material amplificado por PCR se realiza computacionalmente, y representa un factor limitante en el ensayo. Controles y otras consideraciones importantes incluyen el uso de un anticuerpo de isotipo, así como la evaluación de una región de control del ADN genómico, tal como una región intergénica predicho no estar obligado por la proteína en estudio (o no anticipado para mostrar los cambios en virtud las condiciones experimentales). Además, una curva estándar de material de entrada para cada muestra de chip se utiliza para obtener los niveles máximos absolutos de enriquecimiento en el material experimental. El uso de las curvas de calibración ayuda a tener en cuenta las diferencias entre los conjuntos de cebadores, sin importar lo cuidadosamente que están diseñados, así como la eficiencia de diferentescias en todo el rango de concentraciones de la plantilla para un solo grupo de cebadores. Nuestro protocolo es diferente a otros que están disponibles en el 5-8 que cubrimos ampliamente la fase posterior análisis.

La inmunoprecipitación de cromatina (CHIP) de protocolo que aquí se presenta controles de las diferencias, en su caso, de la cantidad de ADN utilizado en la PCR mediante el uso de un par de cebadores que amplifica una región no unida del genoma, lo que sirve como un &quot;control de carga&quot;. En el ejemplo mostrado en la Figura 3, se ha utilizado la región de codificación del gen Actb ratón como una región no unida a nuestra proteína de interés y el sitio de unión del factor de tra...

Watch this Scientific Journal Video about Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass at JoVE.com

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Se describe un método robusto para la inmunoprecipitación de la cromatina utilizando células T primarias. El método se basa en los enfoques estándar, pero utiliza un conjunto específico de condiciones y reactivos que mejoran la eficiencia de unas cantidades limitadas de células. Es importante destacar que, se presenta una descripción detallada de la fase de análisis de datos.

Immunoprecipitation cromatina eficiente utilizando limitación de las cantidades de biomasa

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

efficient-chromatin-immunoprecipitation-using-limiting-amounts

Research

JoVE Journal

Biology

14.2K vistas.  University of Utah School of Medicine. Se describe un método robusto para la inmunoprecipitación de la cromatina utilizando células T primarias. El método se basa en los enfoques estándar, pero utiliza un conjunto específico de condiciones y reactivos que mejoran la eficiencia de unas cantidades limitadas de células. Es importante destacar que, se presenta una descripción detallada de la fase de análisis de datos.

Video: Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Investigación

Biología

Methylated DNA Immunoprecipitation

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

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

Inmunoprecipitación de ADN metilado

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

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Inmunoprecipitación de cromatina (CHIP) para el ensayo dinámico de modificación de las histonas en la expresión génica activado en las células humanas

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Ensayo de inmunoprecipitación de la cromatina de los genes específicos de tejido utilizando etapa temprana de embriones de ratón

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Cuantificar la frecuencia de tumores se propagan las células usando trasplante de células limitación de dilución en el pez cebra singénico

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Inmunoprecipitación de cromatina (CHIP), utilizando Drosophila Tejido

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Generación de Plantilla ADN Immunoprecipitation cromatina alta calidad para la secuenciación de alto rendimiento (chip-ss)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Un método eficaz para Cuantitativo, Análisis de celda única de la cromatina modificación y Arquitectura Nuclear en Todo el montaje en Óvulos<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of 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.

Identificación de proteínas compañeros de interacción en células de mamífero utilizando SILAC-inmunoprecipitación cuantitativos Proteómica

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

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.

Ensayo de inmunoprecipitación de la cromatina para la Identificación de Arabidopsis proteína-DNA<em&gt; En Vivo</em

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