Desarrollo del método de NU-ELISA fue apoyado por el NIH subvención RO1AG023687.

1. Cultivo de células mamíferas NU-ELISA se puede realizar en cualquier tipo de células de mamíferos que pueden ser cultivadas en la cultura. Nosotros preferimos preparar moderada a grandes cantidades de células, de modo que los nucleosomas se pueden aislar en cantidad suficiente para preparar varios idéntica carga placas de ELISA, lo que permite ensayos con varios anticuerpos diferentes (Abs). Las escalas de la cultura después de ofrecer gran cantidad de material: Para que las células madre de ratón embyronic, crecen uno o dos placas de 15 cm de células sin células alimentadoras. Para los fibroblastos, crecer 5 a 10 cm 15 placas a la confluencia. 2. El aislamiento de los núcleos Nota: Todos los pasos están en el hielo con el pre-enfriado tampones, excepto lo indicado. <ol><li> Trypsinize células con 3 ml de tripsina por placa, combinar y agregar 20 ml de helado de PBS / butirato. Centrifugar a 1000 rpm durante 5 min. </li><li> Resuspender las células en 10 ml de PBS / butirato y centrifugar a 1000 rpm durante 5 min. </li><li> Resuspender en 4 ml de tampón de lisis con inhibidores de la proteasa (Sigma-Aldrich P-8340). Dounce homogeneizar 20 golpes con mortero tipo B en el hielo. </li><li> Centrifugar a 2000 g durante 10 min a 4 ° C. (El sobrenadante contiene el citoplasma, que no es necesario para este protocolo, pero se pueden guardar para otros usos si se desea). </li><li> Resuspender el precipitado en 2 ml de hielo frío tampón de lavado C (con inhibidores de la proteasa). </li><li> Capa del material resuspendido en 5 ml colchón de sacarosa al 30% y centrifugar a 2400g durante 5 minutos en un rotor basculante. Los núcleos se migran a través de cojín, y los escombros se mantiene en la interfaz. </li><li> Eliminar todo el volumen de líquido con cuidado y volver a suspender los núcleos de hielo de 250 l de tampón de lavado en frío C + inhibidores de la proteasa. </li></ol> 3. Aislamiento de los nucleosomas in situ por micrococcal nucleasa (MNase) Digestión Utilizamos un procedimiento en el cual se digiere la cromatina in situ dentro de los núcleos mediante la infusión con MNase, seguido de un tratamiento hipotónico para conducir sin nucleosomas intacta en el sobrenadante. <ol><li> Añadir 3 l 0,1 M CaCl 2 a la muestra. Puesto en 37 ° C del bloque de calor. Permiten suponer 37 ° C de temperatura. </li><li> Añadir 2 unidades MNase (micrococcal nucleasa, Sigma-Aldrich, disuelto en 2 units/10μl en tampón MNase), a continuación, se incuba durante a 37 ° C durante 12 minutos, mezclando frecuentemente utilizando una punta de pipeta. </li><li> Añadir 6 l de 0,5 M Na-EDTA, pH 8,0 para detener la reacción. Poner en hielo. </li><li> Centrifugar a 2000 g durante 4 minutos, desechar el sobrenadante. Resuspender el precipitado en 300 l 0,2 mM Na-EDTA. Guárdelo en hielo durante 1 h con pipeta suave ocasional. (Estas condiciones hipotónicas liberar a los nucleosomas libres de los núcleos). </li><li> Centrifugar a 3000 g durante 4 min a 4 ° C. Guardar el sobrenadante, que contiene nucleosomas libre, en el hielo. </li><li> Resuspender el precipitado de nuevo con 300 l 0,2 mM Na-EDTA. Guárdelo en hielo durante 1 h con pipeta suave ocasional. </li><li> Centrifugar a 3000 g de 4 minutos a 4 ° C. Conserve el sobrenadante y se combinan con el primer sobrenadante del paso 5. Las preparaciones resultantes mononucleosome puede alícuotas y almacenados a -80 ° C. </li></ol> Nota: La cantidad de cromatina puede ser cuantificada por la crudeza de medir la absorbancia a 260 nm de una muestra de 10 l añadido a 990 l de agua. A 260 = 10 (después de ajustar por la dilución 1 / 100) corresponde a cerca de 1mg/ml de la cromatina. Además, durante el procedimiento anterior, las muestras pequeñas pueden ser retenidos y luego se analiza su contenido de ADN para controlar la calidad y el alcance de las digestiones MNase, que principalmente debe contener ADN de 146 pb de longitud. Escala es un indicativo de la digestión incompleta MNase. Nota: la figura 1 contiene un resumen diagramado de aislamiento nuclear y las medidas digestiones MNase. 4. Nucleosoma-ELISA (NU-ELISA) Detectamos PTM en las fracciones que contengan especies nativas nuclesosomes intactos que se han inmovilizado en placas de 96 pocillos de microtitulación de ELISA. Para cada muestra, se hace una serie de dos diluciones, y estos están cubiertos en los pozos por triplicado. <ol><li> Escudo MaxiSorp la noche a 4 ° C con 50 l / pocillo de los nucleosomas diluido en tampón de recubrimiento. Placas Sugiere diluciones en serie de los nucleosomas se preparan a partir de la fila superior de la fila inferior mediante la adición de 0,1 mg, 0,05 mg, 0.025 mg, 0,0125 g, mg 0,00625, 0,00313 g, mg 0,00156, con tampón de recubrimiento sólo (0 cromatina mg) en la parte inferior fila. (Nota: cubiertas de la placa son necesarias para este paso.) </li><li> Por la mañana, lavar los platos cuatro veces con 200 l / pocillo PBS/0.5% Tween-20 a temperatura ambiente (TA) para un total combinado de 10 minutos con un lavador de placas. </li><li> Bloque 1 hora a temperatura ambiente con 100 l / pocillo PBS/0.05% Tween-20 / 5% de BSA. </li><li> Retire el amortiguador de bloqueo agitando la placa invertida rápidamentesobre un fregadero. Cubra y guarde las placas a -20 ° C, o ir directamente al siguiente paso. </li><li> Añadir 1 ° Abs en un volumen de 50 l / pocillo diluido 1:1000 (o según sea necesario) en PBS/0.05% Tween-20 / 5% de BSA, se incuban a temperatura ambiente durante 1 hora en un rotor. </li><li> Lavar las placas 4 veces con 200 l / pocillo PBS/0.5% Tween-20 a temperatura ambiente y por un total combinado de 10 minutos en un lavador de placas. </li><li> Añadir peroxidasa de rábano picante (HRP) y conjugado con 2 º Abs, diluido 1:5000 (o según sea necesario) en PBS/0.05% Tween-20 / 5% de BSA a temperatura ambiente durante una hora en un rotor. </li><li> Lavar las placas 4 veces con 200 l / pocillo PBS/0.5% Tween-20. Cada lavado es a temperatura ambiente y por un total de 10 minutos con un lavador de placas. </li><li> Desarrollar las placas mediante la adición de 50 l de sustrato TMB a cada pocillo durante 10 minutos a temperatura ambiente. Detener la reacción añadiendo 50 l / pocillo de H 2 N 2 SO 4 y leer las placas de 450 nm. (Consejo: Se centrifuga a 1500 rpm durante 2 minutos con un rotor de placas para disipar cualquier burbuja de aire en los pozos antes de absorbancias de lectura). </li><li> Exportación de las lecturas de los archivos de hoja de cálculo para el análisis cuantitativo y estadístico. </li></ol> Reactivos <table border="1"><tbody><tr><td> PBS / butirato </td></tr><tr><td> 135 mM NaCl </td></tr><tr><td> 2,5 mM de KCl </td></tr><tr><td> 8 mM Na 2 HPO 4 </td></tr><tr><td> 1,5 mM KH 2 PO 4 </td></tr><tr><td> 10 mM Na-butirato </td></tr></tbody></table><table border="1"><tbody><tr><td> Tampón de lisis </td></tr><tr><td> 250 mM sacarosa </td></tr><tr><td> 10 mM Tris-HCl, pH 7,4 </td></tr><tr><td> 10 mM Na-butirato </td></tr><tr><td> 4 mM de MgCl 2 </td></tr><tr><td> 0,1% Triton X-100 </td></tr></tbody></table><table border="1"><tbody><tr><td> El tampón de lavado C </td></tr><tr><td> 250 mM sacarosa </td></tr><tr><td> 10 mM Tris-HCl, pH 7,4 </td></tr><tr><td> 10 mM Na-butirato </td></tr><tr><td> 4 mM de MgCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Sacarosa cojín </td></tr><tr><td> 30% (w / v) de sacarosa en tampón de lavado C </td></tr></tbody></table><table border="1"><tbody><tr><td> Microccocal nucleasa Buffer </td></tr><tr><td> 5 mM NaPO 4 tampón, pH 7,0 </td></tr><tr><td> 0,025 mM CaCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Revestimiento de amortiguación </td></tr><tr><td> Solución de 80 ml ​​de A + B + 170ml de solución 250ml dH 2 O </td></tr><tr><td> Solución A: 0,2 M Na 2 CO 3 </td></tr><tr><td> Solución B: 0,2 M NaHCO 3 </td></tr></tbody></table> 5. Resultados representante Utilizando el método ELISA-NU, una serie de varias placas idénticas están preparados que se pueden cargar con la cromatina preparados en forma de mononucleosomes. Por lo general preparar una serie de placas cargadas de idéntico modo que cada uno se puede probar con diferentes anti-PTM anticuerpos específicos (Abs). Además, siempre preparar un plato que se puede probar con un Ab que detecta las histonas, independientemente de su estado de modificación total control de la cromatina de carga. Este control es esencial para luego comparar cuantitativamente el contenido de los nucleosomas PTM preparado a partir de diferentes muestras. Para cuantificar los niveles de PTM dentro de una muestra de la cromatina dado que corregir los valores de absorbancia primas que utilizan este enfoque: en primer lugar, se le resta cualquier señal de fondo con los valores obtenidos de los pozos de control que no contiene la cromatina (de fondo suele ser insignificante). A continuación, estandarizar PTM señales específicas de NU-ELISA los resultados obtenidos a partir de una placa idéntica carga que se ensayó con un Ab que detecta los nucleosomas independiente de su estado de modificación. (Hemos utilizado Abs policlonales específicos para las histonas H2A y H2B para este propósito). A continuación, determinar los medios y las varianzas para cada dilución de la cromatina, y el uso de los datos obtenidos de la parte lineal de la prueba de ELISA. Ejemplos detallados de NU-ELISA análisis matemáticos y estadísticos han sido reportados previamente 4 <img src="/files/ftp_upload/2593/2593fig1.jpg" alt="Figura 1"/> Figura 1. Esquemática diagam de NU-ELISA pasos. Las células de mamíferos se cosechan A., B. Los núcleos están separados de las células por Dounce homogenization, C. Interrupción células se cargan en la parte superior de un colchón de sacarosa al 30% w / v, D. Después de la centrifugación, los núcleos se depositan en el sedimento, E, F. cromatina son digeridos in situ a mononucleomes predominantemente por MNase, G, H , yo. mononucleosomes solubles son extraídos por tratamiento hipotónico y centrifugación repetida de material nuclear residual. Mononucleosomes J. partir de diferentes muestras (con la etiqueta samp. 1 y 2 en este caso) están recubiertos en pocillos de microtitulación en serie de forma idéntica carga placas e interrogado en PTM-específicas Abs Abs y PTM-independiente para determinar la carga total de la cromatina. Representación K. de los diferentes niveles de detección de señales en dos muestras que difieren en su contenido PTM, sin embargo, tienen un contenido similar de la cromatina en general, a juzgar por los anti-H2B inmunoreactividad.

<ol>
	<li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+and+an+epigenetic+code.">Histone acetylation and an epigenetic code.</a> Bioessays. 22, 836-845 (2000).</li><li>Dai, B., Rasmussen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epiproteomic+signatures+distinguish+embryonic+stem+cells+from+differentiated+cells.">Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells.</a> Stem Cells. 25, 2567-2574 (2007).</li><li>Tanasijevic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+accumulation+of+epigen+etic+heterogeneity+during+human+ES+cell+culture.">Progressive accumulation of epigen etic heterogeneity during human ES cell culture.</a> Epigenetics. 4, 330-338 (2009).</li><li>Thomas, J. O., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+fractionation+of+chromatin+and+linker+histones.">Isolation and fractionation of chromatin and linker histones.</a> Chromatin: a practical approach. , 12-14 (1998).</li><li>Thorne, A. W., Cary, P. D., Crane-Robinson, C., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+separation+of+core+histones+and+non-histone+chromosomal+proteins.">Extraction and separation of core histones and non-histone chromosomal proteins.</a> Chromatin: a practical approach. , 38-39 (1998).</li></ol>

NU-ELISA proporciona un método para determinar el estado global de la PTM nucleosoma presente dentro de un tipo de célula particular. NU-ELISA estudios han demostrado que la modificación global de los estados nucleosoma difieren en las comparaciones de tipos de células divergentes 4. Además, NU-ELISA perfiles PTM cambiar cuando las células están expuestas a los agentes moduladores epigenéticos como la tricostatina A o cuando el ratón las células madre se diferencian embyronic 4. El método ha sido aplicado con éxito para estudiar la cromatina de células madre embrionarias humanas 5. Es importante tener en cuenta la NU-ELISA, en su forma actual, sólo puede detectar la firma del PTM compuesto presente en la suma total de la epigenoma celular. Esto difiere notablemente de todo el genoma inmunoprecipitación de cromatina, que puede determinar la distribución de un PTM específicos a través de loci genéticos específicos. Los primeros pasos de los procedimientos de NU-ELISA son una adaptación de los métodos anteriores para aislar mononucleosomes de los núcleos de mamíferos 6,7. Estos métodos adaptados proporcionan una manera conveniente de obtener alta calidad mononucleosomes intacta a partir de células de mamíferos, y las fracciones resultantes se completa en su contenido de la cromatina, pero contienen una gran cantidad de materiales nucleares adicionales. Desde Abs específicos se utilizan, la contaminación no nucleosomal material es bien tolerado en el ensayo de NU-ELISA, a diferencia de la masa de las especificaciones enfoques que requieren una mayor pureza. Sin embargo, NU-ELISA es más sensible y cuantitativo de Western Blot 4, proporcionando así una buena alternativa a los westerns y espectrómetro de masas para la detección de PTM nucleosomal.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Micrococcal Nucleae </td><td> Sigma </td><td> N3755 </td><td> Hacer alícuotas de 2 buffer U/10μl, almacenadas a -20 ° C </td></tr><tr><td> Nunc MaxiSorp placas de microtitulación </td><td> Pescador </td><td> 12-565-135 </td><td> Cubre la placa adhesiva también se pueden solicitar a través de Fisher </td></tr><tr><td> Lavadora automática de placas </td><td></td><td></td><td></td></tr><tr><td> Paso 1-TMB-ELISA sustrato </td><td> Atravesar </td><td> 34028 </td><td> Almacenar a 4 ° C antes de calentar a TA antes de su uso o como se indica en la hoja de especificaciones </td></tr><tr><td> Lector de Placas de microtitulación </td><td> Bio-Rad lector de microplacas (modelo 680) </td><td></td><td> La mayoría de los modelos colorimétricos lector de placas son adecuadas </td></tr></tbody></table>

detection of post-translational modifications on native intact nucleosomes by elisa

El genoma de los eucariotas existe como cromatina que contiene el ADN y las proteínas. La unidad fundamental de la cromatina es el nucleosoma, que contiene 146 pares de bases del ADN asociado con dos cada una de las histonas H2A, H2B, H3, H4 y 1. Las colas N-terminal de las histonas son ricas en lisina y arginina y se modifican después de la transcripción por acetilación, metilación y otras modificaciones post-traduccionales (PTM). La configuración de la polilla de los nucleosomas pueden afectar la actividad transcripcional del ADN asociadas, proporcionando así un modo de regulación de los genes epigenéticos que es de naturaleza 2,3. Hemos desarrollado un método llamado ELISA nucleosoma (NU-ELISA) para determinar cuantitativamente las firmas globales PTM de los nucleosomas extraído de células. NU-ELISA es más sensible y cuantitativo de Western Blot, y es útil para interrogar el estado epiproteomic de tipos de células específicas. Este artículo de la revista video muestra a los procedimientos detallados para llevar a cabo análisis de NU-ELISA.

Watch this Scientific Journal Video about Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA at JoVE.com

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Nucleosoma ELISA (NU-ELISA) es un método sensible y cuantitativo para detectar los patrones globales de modificaciones post-traduccionales en la preparación de los nucleosomas nativos, intacto. Estas modificaciones incluyen metilaciones, acetilaciones y fosforilaciones de residuos de histonas específicas de aminoácidos, y por lo tanto, NU-ELISA proporciona un ensayo global de proteómica de los estados de la modificación general de la cromatina de los tipos de células específicas.

La detección de modificaciones post-traduccionales de los nativos intactos nucleosomas por ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

detection-post-translational-modifications-on-native-intact

Research

JoVE Journal

Biology

17.2K vistas.  Stanford University. Nucleosoma ELISA (NU-ELISA) es un método sensible y cuantitativo para detectar los patrones globales de modificaciones post-traduccionales en la preparación de los nucleosomas nativos, intacto. Estas modificaciones incluyen metilaciones, acetilaciones y fosforilaciones de residuos de histonas específicas de aminoácidos, y por lo tanto, NU-ELISA proporciona un ensayo global de proteómica de los estados de la modificación general de la cromatina de los tipos de célul...

Video: Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

La detección de metaloproteinasas de la matriz funcional por Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Investigación

Biología

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

El análisis de la función de las pequeñas GTPasas de la microinyección de plásmidos en células epiteliales polarizadas

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

La detección de modificaciones de las histonas en hojas de las plantas

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Florecimiento extracción de proteínas de levadura y purificación para el Estudio de la función, las interacciones y modificaciones post-traduccionales

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Identificación de modificaciones post-traduccionales de la proteína vegetal Complejos

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

El aislamiento de regiones genómicas específicas e identificación de moléculas asociadas por enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

En Detección de la cara del óxido nítrico y superóxido en la capa endotelial de las arterias intactas

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

De alto rendimiento CRISPR vector de la construcción y caracterización de las modificaciones del ADN mediante la generación de raíces peludas tomate

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Flujo de trabajo completo para el análisis de las histonas modificaciones post-traduccionales El uso de espectrometría de masas de abajo hacia arriba: A partir de la histona Extracción de Análisis de Datos

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Comparación de los métodos de tabaco Protein Removal célula huésped mediante escaldado plantas intactas o por tratamiento térmico de los extractos

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...