Name: Captura de roturas de sitios frágiles comunes mediante γH2A nativo. X ChIP (Video) | JoVE Journal
Uploaded: 2025-01-24T14:20:00.000+00:00
Duration: 9 min 46 s
Description: Aprende a detectar roturas en sitios frágiles comunes mediante ChIP nativo de γH2A.X, un método rápido y reproducible para estudiar la inestabilidad genómica en cáncer. Esta técnica reduce el tiempo y el trabajo comparado con métodos tradicionales.

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<li>Durkin, S. G., Glover, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosome+fragile+sites%5BTitle%5D)+AND+%22Ann+Rev+Genetics%22%5BJournal%5D)">Chromosome fragile sites.</a> Ann Rev Genetics. 41, 169-192 (2007).</li>
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<li>Miyamoto, R., Yokoyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+fractionation-assisted+native+ChIP+%28fanChIP%29+to+capture+protein-protein%2FDNA+interactions+on+chromatin%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Protocol for fractionation-assisted native ChIP (fanChIP) to capture protein-protein/DNA interactions on chromatin.</a> STAR Protoc. 2 (2), 100404(2021).</li>
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<li>Lu, X., Parvathaneni, S., Hara, T., Lal, A., Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replication+stress+induces+specific+enrichment+of+RECQ1+at+common+fragile+sites+FRA3B+and+FRA16D%5BTitle%5D)+AND+%22Mol+Cancer%22%5BJournal%5D)">Replication stress induces specific enrichment of RECQ1 at common fragile sites FRA3B and FRA16D.</a> Mol Cancer. 12, 29(2013).</li>
<li>Ruijter, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amplification+efficiency%3A+linking+baseline+and+bias+in+the+analysis+of+quantitative+PCR+data%5BTitle%5D)+AND+%22Nucl+Acids+Res%22%5BJournal%5D)">Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.</a> Nucl Acids Res. 37 (6), e45(2009).</li>
<li>Lyu, X., Chastain, M., Chai, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+mapping+and+profiling+of+%CE%B3H2AX+binding+hotspots+in+response+to+different+replication+stress+inducers%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Genome-wide mapping and profiling of γH2AX binding hotspots in response to different replication stress inducers.</a> BMC Genomics. 20, 579(2019).</li>
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</ol>

<table><tbody><tr><td>0.2 &micro; Membrana de nitrocelulosa M</td><td>Amersham</td><td>10600011</td><td></td></tr><tr><td>Actina B</td><td>Tecnología de proteínas</td><td>20536-1-AP</td><td></td></tr><tr><td>Afidicolina</td><td>MedChemExpress</td><td>HY-N6733</td><td></td></tr><tr><td>Perlas magnéticas de proteína A / G de grado ChIP</td><td>ThermoFisher</td><td>26162</td><td></td></tr><tr><td>Sustrato Clarity Western ECL</td><td>Bio-Rad</td><td>#1705061</td><td></td></tr><tr><td>Glucógeno, grado de biología molecular</td><td>ThermoFisher</td><td>Gato. No.&nbsp; R0561</td><td></td></tr><tr><td>Anticuerpo secundario conjugado con HRP&nbsp;</td><td>Tecnología de proteínas</td><td>SA00001-2</td><td></td></tr><tr><td>hidroxiurea</td><td>MedChemExpress</td><td>HY-B0313</td><td></td></tr><tr><td>Nucleasa microcócica&nbsp;</td><td>NEB</td><td>M0247S</td><td></td></tr><tr><td>IgG normal</td><td>Santa Cruz</td><td>SC-2025</td><td></td></tr><tr><td>Taq Universal SYBR Green Supermix&nbsp;</td><td>BioRad</td><td>1725120</td><td></td></tr><tr><td>&gamma; H2A. Anticuerpo X&nbsp; (para ChIP)</td><td>Sigma-Aldrich</td><td>05-636</td><td></td></tr><tr><td>&gamma; H2A. Anticuerpo X (para WB)</td><td>Señalización celular</td><td>#25955</td><td></td></tr></tbody></table>

captura de roturas de sitios frágiles comunes mediante γh2a nativo. x chip

Capturing Common Fragile Site Breaks by Native &#947;H2A.X ChIP

El tamaño de los fragmentos de cromatina es crucial para el éxito de la ChIP nativa, ya que afecta directamente a la accesibilidad de las regiones de ADN para la unión de anticuerpos. Para determinar la concentración óptima de MNasa para la fragmentación de la cromatina, preparamos una serie de tubos de microcentrífuga que contenían concentraciones variables de MNasa (es decir, 0,0625 U, 0,125 U, 0,25 U, 0,5 U, 1 U, 2 U, 4 U, 8 U por reacción) y 40 μL de núcleos aislados. Cada reacción se incubó a 37 °C durante 5 minutos...

Captura de roturas de sitios frágiles comunes mediante γH2A nativo. X ChIP

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be ...

capturing-common-fragile-site-breaks-by-native-h2ax-chip

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JoVE Journal

Biochemistry

Bioquímica

Se describe aquí la operación de un dispositivo de microfluidos que permite imágenes microscópicas continua y de alta resolución de las células de levadura en ciernes individuales durante su completa replicativa y / o cronológico vida útil.

Continua observación microscópica de alta resolución de replicativa envejecimiento en la levadura en ciernes

En este artículo se presenta un chip de microfluidos para el análisis de células individuales. Permite la cuantificación de las proteínas intracelulares, enzimas, cofactores, y segundos mensajeros por medio de ensayos fluorescentes o inmunoensayos.

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La ruptura dieléctrica dependiente del tiempo (TDDB) en las pilas de interconexión en chip es uno de los mecanismos de fallo más críticos para los dispositivos microelectrónicos. Este artículo demuestra el procedimiento de un experimento in situ de TDDB en el microscopio electrónico de transmisión, que abre la posibilidad de estudiar el mecanismo de fallo en productos microelectrónicos.

En Desglose dieléctrica Situ dependiente del tiempo en el microscopio electrónico de transmisión: una posibilidad de comprender el mecanismo de falla en los dispositivos microelectrónicos

Informamos de un protocolo para combinar la metrología atómica del Microscopio de Efecto Túnel de Barrido para el modelado de superficies con la Deposición selectiva de la Capa Atómica y el Grabado de Iones Reactivos. Utilizando un proceso robusto que implica numerosas exposiciones atmosféricas y transporte, se fabrican nanoestructuras 3D con metrología atómica.

La fabricación de nanoestructuras Atómicamente trazable

Aquí presentamos los protocolos experimentales para la observación en tiempo real de una uno mismo-montaje proceso mediante microscopía electrónica de transmisión de líquido celular.

Microscopia electrónica de transmisión de líquido de la célula para el rastreo de autoensamblaje de las nanopartículas

Aquí, presentamos un protocolo que describe la fabricación de elástico, doble canal, órgano chip microfluídico célula cultura dispositivos Recapitulando a nivel órgano funcionalidad in vitro.

Fabricación escalable de elástico, doble canal, Chips microfluídicos órgano

Este artículo describe un protocolo junto con un estudio comparativo de dos técnicas de fabricación microfluídica, a saber, fotolitografía/grabado húmedo/enlace térmico y grabado selectivo inducido por láser (SLE), que son adecuadas para condiciones de alta presión. Estas técnicas constituyen plataformas que permiten la observación directa del flujo de fluidos en medios permeables suplentes y sistemas fracturados en condiciones de depósito.

Técnicas de fabricación microfluídica para pruebas de alta presión de transporte de espuma de CO2 supercrítico supercrítico a microescala en depósitos no convencionales fracturados

Aquí, presentamos un protocolo para realizar experimentos in situ de reacción de gas de célula cerrada TEM mientras se detallan varios métodos de preparación de muestras de uso común.

Realización De Reacciones In Situ De Gases De Células Cerradas En El Microscopio Electrónico De Transmisión

Se proporciona un protocolo para el cultivo y manipulación de tejido embrionario de ratón en un chip microfluídico. Mediante la aplicación de pulsos de moduladores de vía, este sistema se puede utilizar para controlar externamente las oscilaciones de señalización para la investigación funcional de la somitogénesis del ratón.

Un enfoque de microfluídica para la investigación funcional de las oscilaciones de señalización que gobiernan la somitogénesis

Este artículo rastrea el ciclo de vida de un resonador de membrana de nitruro de silicio, desde el diseño hasta la caracterización hasta la fabricación y la fabricación.

Fabricación y caracterización de resonadores de membrana de nitruro de silicio de alta calidad

Un protocolo de sellado novedoso para producir sellos herméticos a base de silicona para membranas de dispositivos de chip de polidimetilsiloxano (PDMS). Destinado a laboratorios, montaje de modelos de chips y desarrollo de plataformas de chips a pequeña escala. Demostramos el protocolo utilizando membranas plásticas y tisulares nativas. Este procedimiento solo requiere PDMS, tolueno, moho, membrana y una cámara de vacío.

Procedimiento accesible de sellado de silicona chip-membrana para una unión flexible y fiable

Aprende a detectar roturas en sitios frágiles comunes mediante ChIP nativo de γH2A.X, un método rápido y reproducible para estudiar la inestabilidad genómica en cáncer. Esta técnica reduce el tiempo y el trabajo comparado con métodos tradicionales.

Captura de roturas de sitios frágiles comunes mediante γH2A nativo. X ChIP

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