Work in the authors' laboratory is supported by a grant from the National Institute of General Medical Sciences (GM41628) and by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.

1. ATP-dependientes Nucleosome Remodelación ensayos Para medir las actividades de remodelación nucleosoma dependiente de ATP, inmunopurificado INO80 o subcomplexes INO80 se incuban con ATP y un sustrato mononucleosomal, que contiene un único nucleosoma posicionado en un extremo de un fragmento de ADN de 216 pb, marcado con P 32. Los productos de reacción se someten después a electroforesis en geles de poli-acrilamida nativos. <ol><li> Para generar el fragmento de ADN marcado con 32P &#39;601&#39;, amplificar de pGEM-3Z-601 4 un fragmento de ADN de 216 pb que contiene una secuencia de 601 el posicionamiento de nucleosomas final ubicado, utilizando los oligonucleótidos 5&#39;-ACAGGATGTATATATCTGACACGTGCCTGG y 5&#39;-AATACTCAAGCTTGGATGCCTGCAG como adelante y atrás primers. <ol><li> Configurar una reacción de PCR 100 l como se describe en la Tabla 1. </li><li> Lleve a cabo las reacciones de PCR en un termociclador con el siguiente programa: 1 min a 96 ° C, followed por 45 segundos a 94 ° C, 30 segundos a 57 ° C, 60 segundos a 72 ° C durante 30 ciclos; terminando con 7 min a 72 ° C. </li><li> Después de que se terminó la reacción de PCR, eliminar los nucleótidos no incorporados haciendo pasar los productos de reacción dos veces a través de columnas de centrifugado sin nucleasa. </li><li> Ejecutar 5 l de producto de PCR purificado en un gel de agarosa al 1,2% para confirmar que la reacción de PCR generó el fragmento de ADN de ~ 216 pb deseado. </li><li> Diluir 5 l de producto de PCR purificado de 20 veces, y medir la concentración de ADN utilizando un espectrofotómetro UV. El rendimiento promedio es de ~ 40 ng / l. </li><li> Medir la radiactividad de 1 l de producto de PCR purificado en un contador de centelleo. Estimar el etiquetado de eficiencia mediante el cálculo de la cpm / ng. Se espera una reacción de marcado con éxito para producir ~ 15.000 cpm / ng de fragmento de ADN de 601. </li></ol></li><li> Transferencia de células HeLa nucleosomas en el ADN marcado 601 utilizando un método de dilución en serie 5. <ol><li> Mezclar 2 pmol de32 P-etiquetados 601 fragmento de ADN con 6 g de nucleosomas Hela (preparado como se describe 5) en 50 l de un tampón que contenía NaCl 1,0 M, Tris-HCl 10, pH 8,0, EDTA 1 mM, PMSF 0,1 mM, y 1 mM TDT y se incuba a 30 ° C durante 30 min. </li><li> Secuencialmente se diluye la mezcla a 0,8 M, 0,6 M y NaCl 0,4 M por dilución con 12,5 l, 20,8 l, y 41,6 l, respectivamente, de 10 mM Tris-HCl (pH 8,0), EDTA 1 mM, PMSF 0,1 mM, y DTT 1 mM, con una incubación de 30 min a 30 ° C después de cada dilución. </li><li> Además se diluye la mezcla a 0,2 M y luego a NaCl 0,1 M mediante la adición de 125 l y luego 250 l de la misma solución tampón que contiene 0,1% de Nonidet P-40, 20% de glicerol, y 200 mg / ml de BSA, con una incubación de 30 min a 30 ° C entre las dos últimas diluciones. Almacenar el sustrato mononucleosome a 4 ° C durante un máximo de 3 meses. </li></ol></li><li> Realizar reacciones nucleosoma deslizamiento dependientes de ATP en un volumen total de 10 l. El reaction componentes se enumeran en la Tabla 2. Puesto que la concentración de NaCl óptima para INO80 actividad de remodelación nucleosoma es de ~ 50 mM de NaCl (resultados no publicados), ajustar la concentración total de NaCl en cada reacción a 50 mM, teniendo en cuenta la cantidad de contenido en NaCl preparaciones de nucleosomas y enzima. <ol><li> Antes de comenzar a configurar los ensayos, emitidos geles poli-acrilamida nativos (18 x 16 cm). Para preparar un solo gel que contiene 5% de acrilamida (acrilamida: bisacrilamida 37.5: 1), TBE 0,5 x (Tris 45 mM borato, EDTA 1 mM), persulfato de amonio 0,01% (APS), y 0,001% de N, N, N &#39;, N &#39;tetrametiletilendiamina (TEMED), mezclar los ingredientes enumerados en la Tabla 3. Deje que el gel se polimerice por lo menos durante 2 horas a RT. </li><li> Mientras tanto, para que se realice cada reacción, se combinan en un tubo de 1.5 ml con silicona previamente enfriado ~ 20 nM INO80 o subcomplejo INO80 (preparada como se describe 3) con una cantidad de tampón EB100 ( <strong&gt; Tabla 4) suficiente para dar un volumen de 4,75 l. Inmediatamente vuelva a congelar las fracciones que contiene INO80-que quedan en hielo seco en polvo o nitrógeno líquido. </li><li> Establecer un cóctel principal con el resto de los ingredientes, la ampliación por un factor de &#39;X&#39; (donde X = número total de reacciones 3). La cantidad de cada ingrediente necesario para una sola reacción de 10 l se enumera en la Tabla 5; la receta debe ampliarse de acuerdo con el número de reacciones a realizar. Mezclar bien tocando el tubo o pipeta hacia arriba y hacia abajo con un Pipetman y hacer girar el tubo durante unos segundos en una microcentrífuga de sobremesa. </li><li> Dispensar 5,25 l de cóctel de maestro a cada uno de los tubos de reacción preparada en el paso 1.3.2. Mezclar bien pipeteando arriba y abajo. Iniciar las reacciones mediante la transferencia de los tubos de reacción a un baño de 30 ° C o bloque de calor de agua y se incuba durante 2 hr. </li><li> Mientras tanto, preparar &#39;la eliminación de mezcla&#39; cóctel que contiene ADN competidory nucleosomas, la ampliación por un factor de X (X = número total de reacciones + 4). La cantidad de cada ingrediente necesario para preparar 1,5 l de la mezcla para la eliminación de una sola reacción se enumera en la Tabla 6; la receta debe ser ampliado en función del número de ensayos a realizar. </li><li> Terminar reacciones mediante la adición de 1,5 l de la mezcla de la eliminación. Mezclar bien, girar hacia abajo, y se incuba a 30 ° C durante 30 min. </li><li> Mientras tanto, pre-ejecutar el gel de poliacrilamida nativo en una unidad de electroforesis vertical en 100 V durante 30 min a 4 ° C, utilizando TBE 0.5x como tampón con una barra de agitación magnética dentro de la cámara inferior para mantener la circulación de tampón constante. </li><li> Para cargar la muestra, añadir 2,5 l de colorante de carga que contiene 3x TBE, 30% de glicerol, 0,25% azul de bromofenol, y 0,25% de xileno cianol FF. Mezclar bien, girar brevemente las muestras, y la carga en el gel usando puntas de carga. </li><li> Correr el gel a 200 V durante 4,5 horas a 4 ° C con circulación de buffer.</li><li> Para detectar la señal, transferir el gel a una pila de dos hojas de papel de filtro. Envuelva el papel de filtro con el gel en la parte superior mediante una envoltura de plástico transparente, y luego exponerlo a una pantalla de fósforo de almacenamiento a 4 ° C durante el tiempo deseado. </li><li> Busque en la pantalla con un sistema de escáner de imágenes de isótopos y analizar los datos utilizando un software adecuado. </li></ol></li></ol> 2. Mononucleosome ensayos de unión Para ensayar la afinidad de unión de un complejo INO80 dado para mononucleosomes, realice un cambio de movilidad electroforética de ensayo (EMSA) utilizando el sustrato mononucleosomal generada en el paso 1.2. <ol><li> Configurar la reacción de mezclas para ensayos de unión como se ha descrito para los ensayos de remodelación de nucleosomas, pero omitir la ATP y la eliminación de la mezcla de las reacciones; incubar a 30 ° C durante 30 min. </li><li> Añadir 2,5 l de colorante de carga a cada mezcla de reacción, y se aplican a un gel de poliacrilamida nativo que contiene 3,5% de acrilamida (acrilamida: bis 37.5: 1), 1% de glicerol, TBE 0,5 x, 0,01% de APS, 0,001% y TEMED. </li><li> El uso de TBE 0.5x como tampón, ejecute el gel a 200 V durante 2,5 horas a 4 ° C con circulación de tampón y se exponga a una pantalla de fósforo de almacenamiento. </li></ol> 3. ADN y Nucleosoma-ATPasa dependiente Ensayos Realizar ensayos de ATPasa en las mezclas de reacción 5 l que contenía 20 mM Tris-HCl (pH 7,5), NaCl 60 mM, 6,6 mM de MgCl2, 0,8 mM EDTA, 0,015% de Nonidet P-40, 2,5% de glicerol, 0,1 mg / ml de BSA, 1 mM DTT, 0,1 mM PMSF, 2 mM ATP, 2 Ci de [α- 32 P] ATP (3000 Ci / mmol). Para cada complejo INO80 o cantidad de complejo INO80 a ensayar establecer tres reacciones paralelas, una memoria intermedia que contiene EB100 para medir ADN o nucleosoma-ATPasa independiente, uno que contiene ADN plásmido circular cerrado (5.000 pb, ~ 30 nM) para medir el ADN ATPasa dependiente, y una que contiene Hela oligonucleosomas (~ 185 nm) para medir-nucleosoma ATPasa dependiente. Configure todas las reacciones en hielo. <ol&gt; <li> Para cada reacción, se combinan 10-50 nm de la INO80 o INO80 subcomplexes inmunopurificados con una cantidad de tampón EB100 suficiente para dar un volumen de 2,2 l en 1,5 ml tubos de microcentrífuga lubricados pre-refrigerados. Inmediatamente vuelva a congelar las fracciones que contiene INO80-en hielo seco en polvo o nitrógeno líquido. </li><li> Establecer un cóctel maestro. La cantidad de cada ingrediente necesario para una sola reacción se enumera en la Tabla 7. Escala la receta por la ampliación por un factor de 3 (X + 2) 1, donde X = el número de preparaciones INO80 a ensayar. </li><li> Para preparar conteniendo buffer &#39;sub-cócteles&#39; solamente, ADN, o nucleosomas, dispensan 2.5 (X + 2) l del cocktail master en tres tubos separados. Añadir 0,3 (X + 2) l de cualquiera de EB100, el ADN plásmido circular cerrado (1,5 g / l), o Hela oligonucleosomas (1,5 g / l) y mezclar bien. </li><li> Prescindir de 2,8 l de la sub-cóctel apropiado para los tubos de reacción que contienen enzimas con base en St.ep 3.1. Pipetear suavemente hacia arriba y hacia abajo para mezclar; evitar la introducción de burbujas. </li><li> Para iniciar reacciones, transferir los tubos de reacción a un bloque de calor a 30 ° C. </li><li> Después de 5, 15, 30, y 60 min de incubación, detectar 0,5 l de cada mezcla de reacción en una cromatografía de polietilenimina celulosa de capa fina (TLC) placa (20 x 10 cm) en una línea recta al menos 1,5 cm del borde inferior . Devolver inmediatamente tubos de reacción a la 30 ° bloque de calor C por lo que múltiples puntos de tiempo se pueden tomar de un solo tubo. Después de las salpicaduras, seque las placas de TLC utilizando un secador de pelo. </li><li> Transferir las placas de TLC a una cámara de vidrio que contiene suficiente fosfato de potasio 0,375 M (pH 3,5) para permitir que la parte inferior 0,5 cm de la placa de TLC se sumergieron en la solución. </li><li> Cubra la cámara, y esperar que el frente de la fase líquida alcanza la parte superior de las placas de TLC. Inmediato secar las placas a fondo con un secador de pelo. </li><li> Exponga las placas de TLC secas a una pantalla de fósforo de almacenamiento a temperatura ambiente.Busque en la pantalla con un sistema de escáner de imágenes de isótopos y determinar la cantidad de sustrato ATP radiactivo y producto ADP. </li><li> Para calcular la cantidad de ATP hidrolizado, multiplique el ATP% hidrolizado por la cantidad de ATP presente en la mezcla de reacción de partida utilizando la siguiente fórmula: ATP hidrolizado pmol = 10 ATP pmol en el inicio de la reacción x [ADP / (ATP + ADP)] </li></ol>

<ol>
	<li>Clapier, C. R., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+chromatin+remodeling+complexes.">The biology of chromatin remodeling complexes.</a> Annual Review of Biochemistry. 78, 273-304 (2009).</li><li>Narlikar, G. J., Sundaramoorthy, R., Owen-Hughes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functions+of+ATP-dependent+chromatin-remodeling+enzymes.">Mechanisms and functions of ATP-dependent chromatin-remodeling enzymes.</a> Cell. 154 (3), 490-503 (2013).</li><li>Chen, L., Ooi, S. K., Conaway, J. W., Conaway, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+purification+of+human+INO80+chromatin+remodeling+complexes+and+subcomplexes.">Generation and purification of human INO80 chromatin remodeling complexes and subcomplexes.</a> , (2013).</li><li>Lowary, P. T., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+DNA+sequence+rules+for+high+affinity+binding+to+histone+octamer+and+sequence-directed+nucleosome+positioning.">New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.</a> J. Mol. Biol. 276 (1), (1006).</li><li>Owen-Hughes, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+nucleosome+disruption+by+ATP-driven+chromatin+remodeling+complexes.">Analysis of nucleosome disruption by ATP-driven chromatin remodeling complexes.</a> Methods Mol. Biol. 119, 319-331 (1999).</li><li>Udugama, M., Sabri, A., Bartholomew, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+INO80+ATP-dependent+chromatin+remodeling+complex+is+a+nucleosome+spacing+factor.">The INO80 ATP-dependent chromatin remodeling complex is a nucleosome spacing factor.</a> Mol. Cell Biol. 31 (4), 662-673 (2011).</li><li>Jin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mammalian+chromatin+remodeling+complex+with+similarities+to+the+yeast+INO80+complex.">A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex.</a> Journal of Biological Chemistry. 280 (50), 41207-41212 (1074).</li><li>Chen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+organization+of+the+human+INO80+chromatin+remodeling+complex:+an+evolutionarily+conserved+core+complex+catalyzes+ATP-dependent+nucleosome+remodeling.">Subunit organization of the human INO80 chromatin remodeling complex: an evolutionarily conserved core complex catalyzes ATP-dependent nucleosome remodeling.</a> Journal of Biological Chemistry. 286 (13), 11283-11289 (2011).</li><li>Hamiche, A., Sandaltzopoulos, R., Gdula, D. A., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP-dependent+histone+octamer+sliding+mediated+by+the+chromatin+remodeling+complex+NURF.">ATP-dependent histone octamer sliding mediated by the chromatin remodeling complex NURF.</a> Cell. 97 (7), 833-842 (1999).</li><li>Polach, K. J., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+enzymes+as+probes+of+nucleosome+stability+and+dynamics.">Restriction enzymes as probes of nucleosome stability and dynamics.</a> Methods Enzymol. 304, 278-298 (1999).</li><li>Anderson, J. D., Thastrom, A., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+access+of+proteins+to+buried+nucleosomal+DNA+target+sites+occurs+via+a+mechanism+that+is+distinct+from+nucleosome+translocation,+Mol.Cell+Biol.">Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation, Mol.Cell Biol.</a> 22 (20), 7147-7157 (2002).</li><li>Saha, A., Wittmeyer, J., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+remodeling+through+directional+DNA+translocation+from+an+internal+nucleosomal+site.">Chromatin remodeling through directional DNA translocation from an internal nucleosomal site.</a> Nature Structural and Molecular Biology. 12 (9), 747-755 (2005).</li><li>Gottschalk, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ADP-ribosyl)ation+directs+recruitment+and+activation+of+an+ATP-dependent+chromatin+remodeler,+Proc.Natl.Acad.Sci.U.S.A.">Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler, Proc.Natl.Acad.Sci.U.S.A.</a> 106 (33), 13770-13774 (2009).</li><li>Clapier, C. R., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+ISWI+involves+inhibitory+modules+antagonized+by+nucleosomal+epitopes.">Regulation of ISWI involves inhibitory modules antagonized by nucleosomal epitopes.</a> Nature. 492 (7428), 280-284 (2012).</li><li>Brune, M., Hunter, J. L., Corrie, J. E. T., Direct Webb, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Measurement+of+Rapid+Inorganic+Phosphate+Release+Using+a+Novel+Fluorescent+Probe+and+Its+Application+to+Actomyosin+Subfragment+1+ATPase,+Biochemistry.">Real-Time Measurement of Rapid Inorganic Phosphate Release Using a Novel Fluorescent Probe and Its Application to Actomyosin Subfragment 1 ATPase, Biochemistry.</a> 33 (27), 8262-8271 (1994).</li><li>Luk, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+histone+replacement+by+SWR1+requires+dual+activation+with+histone+H2A.Z+and+canonical+nucleosome.">Stepwise histone replacement by SWR1 requires dual activation with histone H2A.Z and canonical nucleosome.</a> Cell. 143 (5), 725-736 (2010).</li></ol>

The authors declare that they have no competing financial interests.

Para asegurarse de que la remodelación nucleosoma y actividades de ATPasa se observa en los ensayos dependen de la actividad catalítica de complejos INO80, y no en la contaminación de la remodelación y / o enzimas ATPasa, que nucleosoma ensayo de forma rutinaria la remodelación y la actividad ATPasa de versiones catalíticamente inactivos de Ino80, purificado en en paralelo con el tipo salvaje INO80 utilizando el mismo procedimiento. Una reacción de control negativo que carece de ATP también se debe realizar cuan...

Complejos remodeladores de cromatina familia SNF2 incluyen una SNF2 como ATPasa subunidad central de 1,2. Algunos ATPasas función como SNF2 como enzimas de subunidades individuales, mientras que otros funcionan como la subunidad catalítica de complejos de múltiples subunidades más grandes. Dilucidar los mecanismos moleculares por los cuales cada una de las subunidades de la cromatina complejos remodeladores de contribuir a sus actividades requiere la capacidad de realizar ensayos bioquímicos que diseccionan el proceso de remodelación. Remodelación nucleosoma dependiente de ATP por el complejo INO80 humana y otras enzimas remodelación de la cromatina puede ser concebido como un proceso de múltiples pasos que comienza con la unión de la enzima a la remodelación nucleosomas, seguido por la activación de su ADN y / o nucleosoma-ATPasa dependiente, translocación de la enzima sobre el ADN nucleosomal remodelación, y la eventual reposicionamiento de los nucleosomas 1,2. La comprensión de los detalles moleculares de la ATP-dependiente proceso de remodelación de la cromatina requiere disección de la reacción de remodelación en sus pasos individuales y definición de las contribuciones de las subunidades individuales del complejo de remodelación de la cromatina a cada etapa de la reacción. Tales análisis requieren la capacidad de analizar la remodelación nucleosoma y otras actividades utilizando sustratos moleculares definidos in vitro. En un protocolo JOVE anterior, describimos los procedimientos utilizados para generar complejos remodeladores de cromatina INO80 y subcomplexes con composiciones de subunidades definidas 3. A continuación, presentamos tres ensayos bioquímicos que permiten el análisis cuantitativo de la unión, ADN y-nucleosoma activado ATPasa, y actividades de remodelación de nucleosomas asociados con tales complejos de nucleosomas.

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			<td height="21" style="height:21px;width:359px;">Name of Reagent/Material</td>
			<td style="width:212px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:123px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">P8340</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PCR reaction buffer&#160;</td>
			<td style="width:212px;">Roche Applied Science&#160;</td>
			<td style="width:147px;">11435094001</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roche Taq DNA Polymerase</td>
			<td style="width:212px;">Roche Applied Science&#160;</td>
			<td style="width:147px;">11435094001</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucAway Nuclease-free Spin Columns&#160;</td>
			<td>Ambion</td>
			<td style="width:147px;">Cat. # AM10070</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ultrapure ATP&#160;</td>
			<td>USB/Affymetrix</td>
			<td style="width:147px;">77241 25 UM</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">A9418&#160;</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">N,N,N&#180;,N&#180;-tetramethylethylenediamine (TEMED)</td>
			<td style="width:212px;">Thermo Scientific</td>
			<td style="width:147px;">17919</td>
			<td style="width:123px;">Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40% Acrylamide/Bis 37.5:1</td>
			<td style="width:212px;">Amresco</td>
			<td style="width:147px;">0254-500ML</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicated salmon sperm DNAs&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">27-4565-01</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% ammonium persulfate (APS)</td>
			<td style="width:212px;">Thermo Scientific</td>
			<td style="width:147px;">17874</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">benzonase&#160;</td>
			<td>Novagen</td>
			<td style="width:147px;">Cat. No. 70664</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">[&#945;-32P] ATP (3000 Ci/mmol)</td>
			<td style="width:212px;">PerkinElmer</td>
			<td style="width:147px;">BLU003H250UC</td>
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			<td height="21" style="height:21px;width:359px;">dCTP, [&#945;-32P]- 6000Ci/mmol</td>
			<td style="width:212px;">PerkinElmer</td>
			<td style="width:147px;">BLU013Z250UC</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Equipment</td>
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			<td height="21" style="height:21px;">PCR thermal cycler PTC 200</td>
			<td>MJ Research</td>
			<td style="width:147px;">PTC 200</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoefer vertical electrophoresis unit</td>
			<td style="width:212px;">Hoefer</td>
			<td style="width:147px;">SE600X-15-1.5</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lubricated 1.5ml microcentrifuge tubes&#160;</td>
			<td style="width:212px;">Costar</td>
			<td style="width:147px;">3207</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Storage Phosphor Screen&#160;</td>
			<td>Molecular Dynamics</td>
			<td style="width:147px;">63-0034-79</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3MM filter paper</td>
			<td>Whatman&#160;</td>
			<td style="width:147px;">28458-005</td>
			<td style="width:123px;">VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Typhoon PhosphorImager&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">8600</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageQuant software</td>
			<td>GE Healthcare</td>
			<td style="width:147px;">ver2003.02</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">TLC Glass Plates, PEI-Cellulose F</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">5725-7</td>
			<td style="width:123px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Immobilon-FL Transfer Membrane 7 x 8.4</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">IPFL07810</td>
			<td style="width:123px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:359px;">General purpose survey meter with end-window or pancake GM (Geiger-Mueller) probe</td>
			<td style="width:212px;">Biodex</td>
			<td style="width:147px;">Model 14C</td>
			<td style="width:123px;"></td>
		</tr>
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biochemical assays for analyzing activities of atp-dependent chromatin remodeling enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Las cifras muestran resultados representativos de ensayos bioquímicos utilizados para caracterizar las actividades INO80, incluyendo nucleosoma deslizamiento (Figura 1) y vinculante (Figura 2) y ensayos de ADN o ATPasa ensayos nucleosomas dependiente (Figura 3). El experimento mostrado en la Figura 1 compara la capacidad de los complejos de INO80 intactas purificó a través de FLAG-NIF N ° 2 o FLAG-INO80E y de INO80 subco...

Watch this Scientific Journal Video about Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes at JoVE.com

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Los ensayos bioquímicos para Actividades Analizando de ATP-dependientes remodelación de la cromatina enzimas

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

biochemical-assays-for-analyzing-activities-atp-dependent-chromatin

Research

JoVE Journal

Biology

14.7K vistas.  Stowers Institute for Medical Research. Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Video: Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Un ensayo de la cromatina de tejido cerebral humano

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Investigación

Biología

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Cerebro del pez cebra ventrículo inyección

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Photobleaching ensayos (FRAP y FLIP) para medir la dinámica de la cromatina de proteínas en células vivas madre embrionarias

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

Dos tipos de ensayos para la detección de espermatozoides quimioatracción rana

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

La cromatina Análisis de Interacción con la etiqueta de secuenciación pareadas final (Chia-PET) para el mapeo de la cromatina y la comprensión de las interacciones Reglamento Transcripción

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Extracción de sangre para análisis bioquímicos en adultos de pez cebra

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Extracción de los antígenos tisulares para Ensayos funcionales

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Bioquímica y de Evaluación de Rendimiento microscópica de masa grasa en<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Titulación bioquímica de glucógeno<em&gt; In vitro</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Los ensayos de viabilidad de las células en cultivo

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...