Estamos muy agradecidos a Michela de Martino, Wilfred R. Hagen y Kourosh Honarmand Ebrahimi útil para los debates. Esta labor fue apoyada con una financiación inicial de la Universidad Tecnológica de Delft.

1. Dps Expresión y purificación La obtención de proteína de alta pureza es un primer paso esencial para el ensayo de protección de ADN. La purificación de la proteína Dps se puede realizar en 4 a 5 días. <ol><li> Transformar una cepa deficiente en proteasa de E. coli (tal como BL21 (DE3) pLysS) con un vector pET (tales como pET17) en el que la secuencia que codifica la proteína Dps ha sido clonado. </li><li> Racha de las células transformadas a cabo en placas de agar Luria Broth (LB) que contienen los antibióticos apropiados (tales como ampicilina y cloranfenicol para los ejemplos proporcionados en 1.1). Las placas se incuban durante la noche a 37 ° C. </li><li> Se inocula una sola colonia de la placa en 30 ml de medio LB que contenía los antibióticos apropiados. Incubar toda la noche a 37 ° C con agitación a 200-250 rpm. </li><li> Inocular 2 x 1 L de medio LB que contiene antibióticos apropiados con 10 ml de cada uno de los cultivo de una noche. Crece a 37 ° C, mientras se agita, a OD 600 </sub&gt; de aproximadamente 0,6. (Cultivo células para producción de proteína aumenta OD más alto, sin comprometer notablemente la pureza del producto final.) Inducir la expresión de la AD mediante la adición de IPTG a una concentración de 0,3 mM, y se incuba a 37 ° C durante 3-4 horas mientras se agita. </li><li> Células de la cosecha por centrifugación a 6000 xg durante 15 min. Resuspender los sedimentos de células en 7,5 ml de DEAE tampón A (50 mM de HEPES-KOH, pH 7,5, 100 mM de NaCl, 0,1 mM EDTA) por litro de cultivo celular inducida. Las muestras pueden ser congeladas en nitrógeno líquido y se almacenan a -80 ° C, si se desea. El procedimiento puede entonces continuarse por descongelar las muestras en un baño de agua a 4 ° C. </li><li> Añadir una mezcla de inhibidores de la proteasa (Calbiochem, tales como inhibidores de la proteasa conjunto III, en 0.167 l por ml de suspensión de células) a la suspensión celular para impedir la degradación de AD sobreexpresados. </li><li> Preparar extracto libre de células usando una prensa francesa. Cebe la prensa francesa con 20 ml de DEAE Buffer A (si se utiliza un modelo de flujo continuo), entonces disrupciónt de la muestra dos veces en 20 kpsi. Se centrifuga el lisado a 30.000 xg durante 35 min a 4 ° C para aclarar las partículas insolubles, y guardar el sobrenadante. El sobrenadante puede ser entonces congelada utilizando nitrógeno líquido y se almacenó a -80 ° C, si se desea. </li><li> Se equilibra una columna de 30 ml de DEAE Sepharose CL-6B de DEAE con tampón A, usando una FPLC. Ejecutar tampón a través de la columna a 1-2 ml / min con una presión máxima de 0,2 MPa sobre el fondo. Cargar el extracto libre de células en la columna, y comenzar a recoger el flujo a través de-una vez que la señal de OD 280 comienza a aumentar por encima de línea de base. Lavar la columna con DEAE tampón A hasta que el valor de la DO 280 vuelve a la línea de base, mientras que continuamente recoger el flujo a través. El flujo a través deben contener la mayoría de la proteína Dps, libre de ADN unido. Lavar la columna con 100% de tampón B (50 mM de HEPES-KOH, pH 7,5, 1 M de NaCl, 0,1 mM de EDTA). Este eluido contiene complejos AD-ADN, así como otras proteínas y puede ser desechada. </li><li> Eliminar publicidadcontaminar condicional proteínas a través de precipitación con sulfato amónico. Determinar el volumen combinado de flujo continuo. Lentamente (durante un período de 10-20 min) añadir sulfato de amonio a pequeña grano seco a la saturación de 62% (390 mg por ml de solución) a 4 ° C mientras se agitaba bien. Se agita la mezcla durante un adicional de 20-30 minutos después de la adición de la última porción de sulfato de amonio para asegurar equilibrio completo. Dps permanecerán solubles, mientras que las proteínas contaminantes sin precipitar fuera de la solución. </li><li> Eliminar las proteínas precipitadas por centrifugación a 20.000 xg durante 30 min a 4 ° C y guardar el sobrenadante. </li><li> Poco a poco agregue un sulfato amónico 227 mg adicionales por ml de sobrenadante para alcanzar la saturación del 94% y se agita durante 20 a 30 minutos para asegurar el equilibrio completo. Dps precipitados en esta alta concentración de sal y pueden ser recogidos por centrifugación a 20.000 xg durante 30 min a 4 ° C. AD estarán en el sedimento, el sobrenadante puede ser desechada. El sedimento se puede almacenar a -80 ° C si desired. </li><li> Resuspender el sedimento que contiene AD-resuspensión en tampón (50 mM de HEPES-KOH, pH 7,5, 150 mM de NaCl, 0,1 mM de EDTA). Ajuste el volumen de la muestra de 2,5 ml. </li><li> El cambio de tampón para eliminar el sulfato de amonio mediante el uso de una columna de filtración en gel PD-10. Equilibrar el lecho de gel con 25 ml de tampón de resuspensión. Aplique el 2,5 ml de AD muestra en la parte superior de la columna de PD-10 y deje que penetre en su interior Deseche el flujo continuo. Recoger AD eluyendo con 3,5 ml de tampón de resuspensión. </li><li> Centrifugar el intercambio de tampón de la muestra a 16.000 xg durante 10 min a 4 ° C para eliminar los componentes insolubles, y diluirlo con 6-10 ml de tampón de dilución (50 mM de HEPES-KOH, pH 7,5, EDTA 0,1 mM) para asegurar que el concentración de sal es lo suficientemente bajo como para AD para unirse a la columna de Sepharose SP. En particular, las muestras concentradas de AD pueden llegar a ser menos soluble tras la dilución en un tampón de menor contenido de sal, como se evidencia por la nubosidad de líquido, pero pueden ser completamente resolubilizada por la adición de una pequeña amount de solución 5 M de NaCl (un adicional de 10-20 mM). </li><li> Se equilibra una columna de flujo de 30 ml de SP Sepharose Fast SP con tampón A (50 mM de HEPES-KOH, pH 7,5, 50 mM de NaCl, 0,1 mM de EDTA). Ejecutar tampón a través de la columna a 1,5-2 ml / min con un límite de presión de 0,3 MPa sobre el fondo. Cargar la muestra en la columna. Lavar con SP tampón A hasta que las declaraciones traza OD 280 de línea de base, y desechar el flujo continuo. Ejecutar un gradiente lineal de 150 ml de 0 a 100% de tampón B, mientras que la recogida fracciones de 2 ml. AD se eluyen en un pico agudo en torno al 50% de B, aunque la variación en un 10% es posible. </li><li> Ejecute las fracciones de elución en un gel SDS-PAGE al 15%, y comprobar la contaminación de ADN mediante la medición de los 260/280 OD relación OD usando un espectrofotómetro. El gel sólo debe mostrar una banda de alrededor de 19 kDa, y el OD 260 / OD 280 es típicamente alrededor de 0,7. Piscina para los más puros fracciones de DPS. Utilice una unidad de filtro centrífugo (tal como la unidad de filtración Amicon Ultra) con un10K peso molecular de corte para concentrar las DPS y cambiarlo en un tampón de almacenamiento (50 mM HEPES-KOH, pH 7,5, 50 mM NaCl). Alícuotas de las AD purificados, y congelar en nitrógeno líquido para su almacenamiento a -80 ° C. </li></ol> 2. Ensayo de protección del ADN El ensayo implica múltiples pasos sensibles al tiempo y debe programarse a la segunda para obtener resultados reproducibles. Muchos de los ingredientes y los pasos de pipeteado están involucrados, por lo que una mesa de pipeteado (ver Tabla 1) se recomienda realizar un seguimiento de las medidas y volúmenes. El tiempo total requerido para el ensayo es de no más de 3 horas. <ol><li> Medir 30 ml de agua en un frasco hermético; enjuagar el agua con N2 durante 10 min (utilizando dos agujas de jeringa, una en el fluido, una encima de la misma) para eliminar el oxígeno de la solución. Añadir 0,0168 g FeSO4 • 7H 2 O para el agua para obtener una solución madre 2 mM. Sellar el frasco y enjuague con nitrógeno durante 5 minutos más, luego mezclar. La solución debe serclaro, si cualquier atisbo de amarillo es detectable, preparar una nueva solución de hierro. </li><li> Hacer un 100 mM H 2 O 2 solución (10,87 l de 9.2MH 2 O 2 en 1 ml de agua); mantener en hielo y protegida de la luz, envolviendo en papel de aluminio. Utilice sólo durante 1-2 horas después de la preparación debido a la descomposición espontánea. El stock de peróxido de hidrógeno es similar sujeto a la ruptura, que puede estar parcialmente mejorado por almacenamiento adecuado (en la oscuridad, a 4 º C). Se recomienda la comprobación periódica de la concentración stock de medición de los 240 OD. </li><li> Descongelar una alícuota de AD purificada rápidamente en un baño de agua a temperatura ambiente, y mantener en hielo. Centrifugar durante 10 segundos a 4000 xg en una tapa picocentrifuge mesa para agregados precipitado. Medir la concentración después de la centrifugación usando un espectrofotómetro (DO 280 = 1,547 indica una concentración de 100 AD monómero mu M). </li><li> Diluir el ADN lineal de alta concentración madre usando 12x tampón de reacción (1M MOPS-KOH pH 7,0, NaCl 1 M) a una concentración de 100 ng / l. ADN lineal se utiliza para hacer más fácil cuantificación, pero el ADN plásmido puede ser utilizado también. </li><li> Pipetear 1 l de la mezcla de ADN-tampón en un tubo de PCR. Añadir suficiente agua en la reacción de modo que el volumen final de reacción (menos SDS) será de 12 l. Este importe debe calcularse con antelación utilizando la tabla de pipeteo. Añadir AD a una concentración dodecámero final de 3 mM. Incubar durante 15 min a temperatura ambiente para permitir la AD a se unen al ADN. </li><li> Añadir suficiente mM de solución de FeSO4 2 para llegar a la concentración deseada. Un experimento típico utiliza una gama de concentraciones de 0 a 1 mM FeSO 4. Las concentraciones más altas causarán más extensa degradación del ADN, pero también pueden conducir a la formación de férrico precipita en la mezcla de reacción. Añadir rápidamente 1 l de la solución 2 mM de H 100 O 2 (a concentración final 10 mM), y se incuba a temperatura ambiente durante 5 min para permitir que el FentonMediada por reacción de degradación a tener lugar. </li><li> Añadir 0,8 l de 20% de SDS (dodecil sulfato de sodio). Mezclar e incubar a 85 ° C durante 5 min para alterar los complejos de ADN-AD. El SDS desestabiliza el complejo AD-ADN y previene cambios no deseados de gel para el ADN, lo que mejora la facilidad de cuantificación. Un paso opcional es para detener la reacción por catalíticamente degradar el peróxido de hidrógeno en productos no tóxicos. En las condiciones descritas en este protocolo, no es necesario este paso para obtener una amplia gama de la degradación del ADN. </li><li> Incubar en hielo durante 1 min, añadir colorante de carga, y cargar en un gel de agarosa. Ejecutar el gel, y de la mancha de ADN utilizando bromuro de etidio. El gel debe ser manchado posterior a electroforesis en lugar de antes de la electroforesis, para evitar que el SDS de interferir con la distribución de bromuro de etidio en el gel. </li></ol>

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No tenemos nada que revelar.

El proceso de purificación de la AD que se describe en este artículo es muy robusto. Pureza ha sido consistentemente alto (&gt; 99%), sin otras proteínas aparecen en geles de SDS-PAGE como bandas visibles. A pesar de esto, algunos lotes de AD purificadas parecen tener una actividad nucleasa, como se evidencia por la degradación parcial de ADN cuando se incubó con concentraciones muy altas de AD. Esto podría indicar la presencia de DNases gran actividad a bajas concentraciones que no fuimos capaces de eliminar a tr...

Los organismos aeróbicos deben enfrentarse constantemente con especies reactivas de oxígeno que pueden dañar su ADN, así como otras macromoléculas biológicas cruciales. Una herramienta potente para contrarrestar los efectos tóxicos de daño oxidativo es la proteína de unión al ADN de las células de hambre (DPS). Desde su descubrimiento en 1992 del hambre E. coli cultura 1, AD se ha identificado en más de 300 especies de bacterias y arqueobacterias 2. Regulación al alza masiva de AD durante la fase estacionaria la convierte en la más alta expresión de proteínas nucleoid asociada de E. coli bajo condiciones de hambre 3, 4. Además, DPS ha sido demostrado para preservar tanto la viabilidad bacteriana y la integridad del ADN durante muchas diversas tensiones, entre ellos el hambre, alta concentración de hierro, exposición a la luz UV, choque térmico, y el estrés oxidativo 5, 6. Estructuralmente, DPS libre asociados a un complejo homo-oligómero estable de 12 monómeros, which montar en una cáscara hueco esférico. El ~ 4,5 nm en toda la cavidad interna es accesible para el disolvente a través de poros que permiten el paso de pequeñas moléculas de 7 exterior, y que es capaz de secuestrar metales mineralizados tales como hierro 8. El efecto protector de la AD se deriva de sus varias actividades bioquímicas, que incluyen ADN no específico de unión 1, la actividad ferroxidasa, y almacenamiento de hierro 8. Estudio detallado de las actividades bioquímicas beneficiosos de AD requiere primero su purificación. AD purificación es un procedimiento elaborado, como AD deben estar separados no sólo a partir de otras proteínas, sino también de cualquier ADN unido 7. Nuestro proceso de purificación optimizado utiliza muchas técnicas comunes, que consiste en dos columnas de intercambio iónico y un paso de precipitación con sulfato de amonio. Se necesitan varios cambios de tampón, como AD altamente concentrados pueden precipitar fuera de la solución en condiciones de poca sal. Una vez que la proteína AD ha sido purificada, Que puede aplicarse a ensayos que miden directamente su actividad ferroxidasa 8, la estequiometría de unión a ADN 9, y los mecanismos de fijación de hierro 10. Dps purificada también tiene otras aplicaciones potenciales. La estructura esférica hueca estable de AD se ha utilizado como andamio para el almacenamiento de partículas hidrófobas dentro de la cavidad proteína 11 e incluso como una cámara de reacción para sintetizar nuevas nanopartículas magnéticas 12. La capacidad protectora de AD para mediar los daños debidos a las especies reactivas de oxígeno puede ser clara y demostrada directamente mediante el ensayo de protección de DNA 13, 14. En este procedimiento in vitro, especies de radicales se producen cuando el hierro cataliza H 2 O 2 a través de la degradación química de Fenton. Estos radicales dañan directamente el ADN presente en la reacción y pueden degradar completamente a altas concentraciones. Dos actividades principales Dps pueden tanto contrarrestar directamente los efectos de Fenten la producción de radicales mediada. AD reduce la concentración de hierro catalítico a través de la mineralización, consumiendo el peróxido de hidrógeno disponible en el proceso. Además, AD unión al ADN potencialmente pueden protegerlo físicamente del daño de los radicales y lo condensa en un volumen más pequeño, con menos superficie reactiva. La combinación de estas dos propiedades hace que el ensayo de protección de ADN muy adecuado para el propósito de medir la actividad protectora AD. El ensayo de protección de ADN es muy versátil y se puede utilizar para una variedad de aplicaciones más allá de AD caracterización. Daño de los radicales es una forma común de estrés en las células, y muchas proteínas y productos químicos diferentes se utilizan para contrarrestarlo. El principio general del ensayo, el uso de la integridad del ADN como un marcador para el daño de los radicales, se puede utilizar en combinación con casi cualquier reacción de radicales-productora o agente de contrarresto. Entre otros, el ensayo ha sido utilizado con éxito para determinar las propiedades anti-oxidantesde K. Extracto paniculata para su uso en la industria alimentaria 15, para caracterizar los efectos del ácido úrico en el daño hidroxilo mediación 16, y para obtener nuevos conocimientos sobre la función de la piel de la transcripción proteínas reguladoras 17. A pesar de los numerosos usos del ensayo en los trabajos publicados, se encontró que eran necesarios muchos optimización y solución de problemas, lo que hace que la configuración del ensayo por primera vez un proceso innecesariamente laborioso para muchos investigadores. El protocolo que presentamos en este artículo tiene como objetivo eliminar esta barrera para la entrada.

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

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

El estrés oxidativo es un subproducto inevitable de la vida aeróbica. El oxígeno molecular es esencial para el metabolismo terrestre, sino que también participa en muchas reacciones perjudiciales dentro de organismos vivos. La combinación de metabolismo aeróbico y el hierro, que es otro compuesto vital para la vida, es suficiente para producir radicales a través de la química de Fenton y degradar los componentes celulares. La degradación del ADN es sin duda el proceso más dañino que implica radicales intracelulares, como la reparación del ADN está lejos de ser trivial. El ensayo presentado en este artículo ofrece una técnica cuantitativa para medir y visualizar el efecto de las moléculas y enzimas en el daño del ADN mediada por radicales. El ensayo de protección de ADN es una herramienta sencilla, rápida y robusta para la caracterización in vitro de las propiedades protectoras de las proteínas o productos químicos. Se supone la exposición de ADN a una reacción oxidativa perjudicial y la adición de concentraciones variables del compuesto de interés. La reducción o el aumento de daño en el ADN como una función de la concentración de compuesto a continuación, se visualizaron mediante electroforesis en gel. En este artículo se demuestra la técnica del ensayo de protección de ADN mediante la medición de las propiedades protectoras de la proteína de unión al ADN de las células de hambre (DPS). DPS es un mini-ferritina que es utilizado por más de 300 especies de bacterias para combatir poderosamente estresores ambientales. Aquí presentamos el protocolo de purificación AD y las condiciones de ensayo optimizados para evaluar la protección del ADN por AD.

El proceso de purificación de AD descritos aquí es muy reproducible. AD purificación de acuerdo con el protocolo descrito, usando 2 l de E. cultura coli como punto de partida, típicamente darán 2,5 ml de proteína que contiene AD 12 a concentraciones entre 5 y 12 mM. Tiempos de inducción más largos (4 horas) parecen reducir esta variabilidad. Pureza proteína es constantemente por encima de 99%, como se evidencia por SDS-PAGE geles (Figura 1). El nivel de contaminaci?...

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

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

La proteína de unión al ADN de las células de hambre (DPS) desempeña un papel crucial en la lucha contra el estrés bacteriano. Este artículo aborda la purificación de<em&gt; E. coli</em&gt; AD y el protocolo para un<em&gt; In vitro</em&gt; Ensayo que demuestra la protección AD-mediada de ADN de la degradación por las especies reactivas de oxígeno.

La aplicación de un<em&gt; In vitro</em&gt; DNA ensayo de protección de visualizar Estrés Mediación propiedades de la proteína Dps

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

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

Research

JoVE Journal

Biology

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

11.1K vistas.  Delft University of Technology. La proteína de unión al ADN de las células de hambre (DPS) desempeña un papel crucial en la lucha contra el estrés bacteriano. Este artículo aborda la purificación de E. coli AD y el protocolo para un In vitro Ensayo que demuestra la protección AD-mediada de ADN de la degradación por las especies reactivas de oxígeno.

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

An In vitro FluoroBlok Tumor Invasion Assay

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

An In vitro FluoroBlok Tumor Invasion Assay

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

Un In vitro Tumor FluoroBlok invasión de ensayo

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

Investigación

Biología

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

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

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

Aplicación de métodos de flujo detenido Cinética para investigar el mecanismo de acción de una proteína de reparación del ADN

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

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

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

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

Los ensayos biofísicos investigará las propiedades mecánicas del núcleo celular Interfase: Aplicación del sustrato deformación y manipulación de Microneedle

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

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

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

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

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

A largo plazo Prueba de toxicidad letal con el Crustáceo Artemia franciscana</em

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

In vitro Methylation Assay to Study Protein Arginine Methylation

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

In vitro Methylation Assay to Study Protein Arginine Methylation

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

In vitro metilación de ensayo para estudiar la proteína arginina metilación

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

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

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

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

Ensayo Cometa como una medida indirecta de estrés oxidativo sistémico

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

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

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

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

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

Medición de daños y reparación del ADN en esplenocitos de ratón Después crónica<em&gt; En Vivo</em&gt; La exposición a dosis muy bajas de beta y gamma Radiación

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

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

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

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

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

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 ...

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

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

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

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

La Asamblea y la Aplicación de &#39;Shear Anillos&#39;: A Novel endotelial Modelo de Orbital, unidireccional y periódica flujo de fluido y la tensión de cizallamiento

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

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

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

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

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

La observación y cuantificación de la conducta de apareamiento en el nematodo del pino,<em&gt; Bursaphelenchus xylophilus</em

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