Nos gustaría dar las gracias al laboratorio de Lee, de la Universidad de Minnesota para proporcionar las muestras de tejido de la médula espinal de ratones que fueron utilizados. Este trabajo fue apoyado por el Departamento de Programa de Defensa de ovario de Investigaciones Oncológicas (OCRP) OC093424 a MB, por el Fondo de Investigación del Cáncer y la Comunidad Randy máquina de afeitar de MB y por el cáncer ovárico de Alianza Minnesota (MOCA) a MB. Los financiadores no tenía papel en el diseño del estudio, la recogida y análisis de datos, decisión de publicar o preparación del manuscrito.

1. Lisis Preparación Buffer <ol><li> Disolver la sacarosa en agua desionizada (DI) agua para hacer una solución stock de 2 M. Filtrar una solución de sacarosa 2 M usando un vacío impulsado 0,22 micras de fluoruro de polivinilideno (PVDF) de filtro. </li><li> Disolver ditiotreitol (DTT) en agua DI para hacer una solución madre 500 mM y almacenar en condiciones anaeróbicas. Disolver el cloruro de magnesio (MgCl2) en agua DI para hacer una solución madre 100 mM. Disolver el trifosfato de adenosina (ATP) hidrato disódico en agua DI para hacer una solución madre 50 mM. </li><li> Hacer una solución de Tris a pH 7,4 mediante la disolución de clorhidrato de Trizma en agua DI y ajustando el pH usando hidróxido de sodio (NaOH). </li><li> Combinar porciones de las soluciones madre en agua DI para hacer un tampón de lisis con una concentración de sacarosa de 250 mM, una concentración de DTT de 1 mM, una concentración de MgCl 2 de 5 mM, una concentración de ATP de 1 mM y una concentración de Tris de 50 mM . Por ejemplo, mezclar 535,3 l de tris, 500 l de MgCl2, 1,25 ml de sacarosa, 20 l de la TDT, 200 l de ATP y 7,4947 ml de agua DI para hacer 10 ml de tampón de lisis. Esto se puede utilizar durante aproximadamente 20 experimentos. </li></ol> 2. Las células de cultivo para el Experimento <ol><li> Hacer alícuotas de los medios de comunicación por 30 ml de DMEM + 10% tubo cónico de suero bovino fetal (FBS) en un 50 ml. </li><li> Traslado células M17 o HeLa congelados de almacenamiento de nitrógeno líquido a un vaso pequeño con agua tibia. </li><li> Transferencia de las células para el tubo cónico de 50 ml. En primer lugar, añadir algunos medios de comunicación para el vial de células. A continuación, traslado a las células resuspendidas en 5 seg de descongelación. </li><li> Centrifugar la suspensión celular a 450 xg durante 5 minutos para obtener un sedimento celular. Añadir 20 ml de medio en un matraz T75. </li><li> Retire el papel de las células por medio de succión. Añadir 5 ml de medio fresco al sedimento celular y resuspender. </li><li> Transferir la suspensión 5 ml de células a las 20 ml de medio en el matraz T75 y se incuba a 37 ° C. Change los medios de comunicación cada 3 días. </li></ol> 3. La recolección de la célula <ol><li> Retire los medios de comunicación a través de succión, con cuidado de no tocar las células. </li><li> Lavar las células con 5 ml de solución salina tamponada con fosfato (PBS). Retire PBS utilizando succión. </li><li> Añadir 3 ml de tripsina-ácido etilendiaminotetraacético (EDTA) al matraz T75 e incubar a 37 ° C durante 3 min. </li><li> Añadir 6 ml de medio fresco al matraz y resuspender las células. </li><li> Transferir la suspensión a un tubo cónico de 15 ml y centrifugar a 290 xg durante 5 minutos. </li><li> Eliminar el sobrenadante y añadir 5 ml de PBS. Golpee suavemente la parte inferior del tubo para romper la pastilla. Centrifugar a 290 xg durante 5 min. </li><li> Repita el paso 3.6 de tres veces. </li><li> Retirar PBS y resuspender las células en 1 ml de PBS fresco. Traslado al tubo Eppendorf (1,5 ml). Girar a 290 xg durante 5 min. </li></ol> 4. Lisis Celular <ol><li> Medir el volumen aproximado de la pastilla usando una pipeta unad pesar cuentas de vidrio en una masa: relación en volumen de 1: 1. Añadir dos veces el volumen del sedimento en tampón de lisis. Nota: Añadir el búfer antes de añadir las perlas de vidrio. </li><li> Lisar las células en el tubo de Eppendorf a la máxima agitación durante 30 min a 4 ° C por agitación en una habitación fría. </li><li> Brevemente Centrifugar a 200 xg durante 30 segundos para resolver las cuentas. Recoger el sobrenadante. </li><li> Añadir el mismo volumen de perlas de vidrio como antes para el sobrenadante. </li><li> Vortex una vez más a la máxima agitación durante 30 min a 4 ° C. </li><li> Brevemente Centrifugar a 200 xg durante 30 segundos para resolver las perlas de vidrio. Recoger el sobrenadante. </li><li> Centrifugar a 5030 xg durante 5 min para eliminar los núcleos, membranas y las células intactas. Recoger el sobrenadante, que es el lisado celular. </li></ol> La homogeneización 5. Tejido <ol><li> Hacer una masa: volumen de 1: 9 solución de tejido de la médula espinal de ratón a tampón de lisis. Nota: Este método es aplicable a una variedad de diferirmuestras de tejido ENT. </li><li> Homogeneizar el tejido usando un ajuste de nivel 2 en el homogeneizador durante 30 segundos para asegurar que no hay trozos restantes. </li><li> Haga girar hacia abajo a 5030 xg durante 3 minutos para eliminar los núcleos, membranas y células intactas. </li><li> Recoger el sobrenadante, que es el lisado. </li></ol> La derivación 6. Muestra <ol><li> Realizar un ácido bicinconínico (BCA) en un análisis de placa de 96 pocillos para determinar la concentración de proteína del lisado de tejido usando un lector de placas de 96 pocillos colorimétrico según lo especificado por el protocolo del kit de ensayo de proteínas Pierce BCA. </li><li> Llevar una alícuota correspondiente a 20 g de proteína total a 50 l usando (DI) de agua desionizada. </li><li> Añadir 2 l de 1,35 M de HA-Ub-Vinil sulfona (VS) a la solución y se incuba durante 1 hora a 37 ° C. Esto da como resultado 20 g de lisado a 50 nM de sonda en la mezcla de reacción final. Esto está en gran exceso molar para asegurar el etiquetado de DUBs funcionales. </li><li> YOncubate la muestra en tampón de muestra de Laemmli durante 5 minutos a 95 ° C. Para un 52 l reacción uso volumen 26 l de tampón de muestra 2x, 13 l de tampón de muestra 4x o 8,87 l de tampón de muestra 6x. </li><li> Enfriar en hielo antes de cargar el gel. </li></ol> 7. Western Blot <ol><li> Cargar un 12 pocillos 4-20% de gel-tris glicina con 40 l de la muestra preparada </li><li> Ejecute el gel a 95 V hasta que el frente de iones alcanza el fondo. </li><li> Llevar a cabo una transferencia durante la noche del gel a una membrana de difluoruro de polivinilideno (PVDF). </li><li> Incubar la membrana en la mancha negro amido y escanear el membane para obtener una imagen que muestra la cantidad de proteína en cada carril. </li><li> Destain la membrana y se incuban en 5% de leche en PBS durante 1 hr a bloquear. </li><li> Incubar la membrana en el anticuerpo primario anti-HA a una concentración de 1: 10.000 en leche 5% en PBS, ya sea durante la noche a 4 ° C o durante 4 horas a temperatura ambiente. </li><li> Realizar 3 lavados fo 5 min cada uno en PBS con 0,1% de Tween-20 detergente. </li><li> Incubar la membrana en un ratón peroxidasa de rábano picante a una concentración de 1: 10.000 en leche 5% en PBS durante al menos 2 hr. </li><li> Realizar 3 lavados de 5 minutos cada uno en PBS con 0,1% de Tween-20 detergente. </li><li> Realizar 1 lavado durante 5 min en PBS. </li><li> Incubar la membrana en un reactivo de detección quimioluminiscente durante 10 min y detectar usando un detector quimioluminiscente. Utilice el ajuste en el detector automático de exposición. </li></ol>

<ol>
	<li>Ovaa, H., Kessler, B. M., Rol&#233;n, U., Galardy, P. J., Ploegh, H. L., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-+based+ubiquitin-specific+(USP)+profiling+of+virus-infected+and+malignant+human+cells.">Activity- based ubiquitin-specific (USP) profiling of virus-infected and malignant human cells.</a> Proc Natl Acad Sci USA. 101 (8), 2253-2258 (2004).</li><li>Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+deubiquitination:+Targeting+of+proteins+for+degradation+by+the+proteasome.">Ubiquitination and deubiquitination: Targeting of proteins for degradation by the proteasome.</a> Semin Cell Dev Biol. 11 (3), 141-148 (2000).</li><li>Ziad, M. E., Wilkinson, K. D. <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+proteolysis+by+human+deubiquitinating+enzymes.">Regulation of proteolysis by human deubiquitinating enzymes.</a> Biochim Biophys Acta. 1843 (1), 114-128 (2014).</li><li>Rol&#233;n, U., 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=Activity+Profiling+of+Deubiquitinating+Enzymes+in+Cervical+Carcinoma+Biopsies+and+Cell+Lines.">Activity Profiling of Deubiquitinating Enzymes in Cervical Carcinoma Biopsies and Cell Lines.</a> Mol Carcinog. 45 (4), 260-269 (2006).</li><li>Borodovsky, A., 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=Chemistry-Based+Functional+Proteomics+Reveals+Novel+Members+of+the+Deubiquitinating+Enzyme+Family.">Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family.</a> Chem Biol. 9 (10), 1149-1159 (2002).</li><li>Andersson, F. 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=The+effect+of+Parkinson&#8217;s-disease-associated+mutations+on+the+deubiquitinating+enzyme+UCH-L1.">The effect of Parkinson&#8217;s-disease-associated mutations on the deubiquitinating enzyme UCH-L1.</a> J Mol Biol. 407 (2), 261-272 (2011).</li><li>Poon, W. W., 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=&#946;-Amyloid+(A&#946;)+oligomers+impair+brain-derived+neurotrophic+factor+retrograde+trafficking+by+down-regulating+ubiquitin+C-terminal+hydrolase+UCH-L1.">&#946;-Amyloid (A&#946;) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase UCH-L1.</a> J Biol Chem. 288 (23), 16937-16948 (2013).</li><li>Nijman, S. M., 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=The+deubiquitinating+enzyme+USP1+regulates+the+Fanconi+anemia+pathway.">The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.</a> Mol Cell. 17 (3), 331-339 (2005).</li><li>Stevenson, L. F., Sparks, A., Allende-Vega, N., Xirodimas, D. P., Lane, D. P., Saville, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP2a+regulates+the+p53+pathway+by+targeting+Mdm2.">The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2.</a> EMBO J. 26 (4), 976-986 (2007).</li><li>Coughlin, K., 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=Small-molecule+RA-9+inhibits+proteasome-associated+DUBs+and+ovarian+cancer+in+vitro+and+in+vivo+via+exacerbating+unfolded+protein+responses.">Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.</a> Clin Cancer Res. 20 (12), 3174-3186 (2014).</li><li>Colla, 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=Endoplasmic+Reticulum+Stress+Is+Important+for+the+Manifestations+of+&#945;-Synucleinopathy+In.">Endoplasmic Reticulum Stress Is Important for the Manifestations of &#945;-Synucleinopathy In.</a> Vivo. J Neurosci. 32 (10), 3306-3320 (2012).</li></ol>

Los autores no tienen nada que revelar.

Desde ubiquitinación es una actividad celular fundamental, la comprensión de los mecanismos de regulación podría ser la clave para sacar a la luz los procesos de la enfermedad y la patología. El uso de HA etiquetada sitio dirigido sondas activas derivadas de Ub aquí presentados proporcionan un método sencillo, pero muy aplicable para el estudio de la degradación de proteínas mediada por Ub. Este método es más rápido y menos costoso que el estudio de cada uno de los DUBs individualmente. <p class="jove_co...

El sistema ubiquitina-proteasoma (UPS) sirve como una de las principales vías de degradación en la célula de mamífero. Sustratos con destino a la degradación en el proteasoma etiquetados de forma covalente con polímeros de ubiquitina (Ub) 1. Antes de que el sustrato elegido como diana entra en el proteasoma para la degradación, la etiqueta de poli-ubiquitina debe ser eliminado. Una clase de enzimas conocidas como deubiquitinating enzimas (DUBs) es responsable de la eliminación y el reciclaje de moléculas de ubiquitina 2. Se ha predicho desde el genoma humano que hay cerca de un centenar DUBs que trabajan en la celda 3. Con un gran número de DUBs que controlan los procesos celulares mediados por Ub tal, el estudio de estas enzimas presenta un desafío ya que las técnicas de ARNm no dan información sobre la actividad y el Western Blot sólo da información sobre los niveles de expresión. El uso de la hemaglutinina de la gripe (HA) etiquetada, Ub derivada de sitio dirigido sondas activas permite un mod covalenteficación de los DUBs funcionales y por lo tanto da una visualización directa de la actividad de estas enzimas en un Western blot 4. Las sondas tienen un grupo reactivo tiol C-terminal que sirve como un sustrato suicida para la cisteína residuo sitio activo 5. Con estas sondas, es posible estudiar la actividad y la expresión potencial de muchos DUBs bajo las dos estados patológicos y fisiológicos de la célula. Cambios en la actividad DUB han sido implicados en una amplia gama de condiciones patológicas tales como Parkinson, Alzheimer, anemia y diversos tipos de cáncer 6-10. Esta técnica proporciona una poderosa herramienta para el estudio de la enfermedad. En el presente trabajo, se muestra la aplicación de esta técnica en las células HeLa y M17 que han sido lisadas usando perlas de vidrio. Además, se describe cómo utilizar este método en muestras de tejido de la médula espinal de ratón. La información obtenida de esta técnica se puede utilizar como punto de partida para la identificación dedianas terapéuticas así como el establecimiento de modelos para el estudio de diferentes condiciones de la enfermedad. La verdadera utilidad de esta técnica radica en su capacidad para proporcionar información sobre múltiples DUBs en un único ensayo.

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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PowerGen 125 ( Homogenizer)</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">14-261-02P</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacuum-driven filters .22&#181;M&#160;</td>
			<td style="width:113px;"></td>
			<td style="width:119px;">BPV2210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid washed &#8804;106&#181;m (~140 U.S. sieve)</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">G4649-10G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">S6-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adenosine 5&#39;-triphosphate disodium salt hydrate</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">A26209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma hydrochloride&#160;</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">T3253</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue culture flask 75cm2 w/ filter cup 250 ml 120/ cs</td>
			<td style="width:113px;">Cellstar</td>
			<td style="width:119px;">T-3001-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HI FBS</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">16140-071</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-HA antibody produced in mouse</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">H9658</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ubiquitin vinyl sulfone (HA-tag)</td>
			<td style="width:113px;">Enzo Life Sciences</td>
			<td style="width:119px;">BML-UW0155-0025</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laemmli&#39;s SDS-Sample Buffer (4X, reducing)</td>
			<td style="width:113px;">Boston BioProducts</td>
			<td style="width:119px;">BP-110R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:113px;">ThermoScientific</td>
			<td align="right" style="width:119px;">23225</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples

El sistema ubiquitina-proteasoma ha sido recientemente implicada en diversas patologías que incluyen enfermedades neurodegenerativas y cáncer. A la luz de esto, las técnicas para estudiar el mecanismo de regulación de este sistema son esenciales para elucidar los procesos celulares y moleculares de las enfermedades mencionadas anteriormente. El uso de hemaglutinina derivado sondas ubiquitina descritos en este documento sirve como una valiosa herramienta para el estudio de este sistema. Este documento detalla un método que permite al usuario realizar ensayos que dan una visualización directa de deubiquitinating actividad de la enzima. Enzimas deubiquitinating control de la degradación proteasomal y comparten homología funcional a sus sitios activos, que permite al usuario investigar la actividad de múltiples enzimas en un ensayo. Los lisados ​​se obtienen a través suave ruptura celular mecánica y se incubaron con sitio activo sondas dirigidas. Enzimas funcionales etiquetados con las sondas mientras enzimas inactivas permanecen sin unir. Por plazoNing este ensayo, el usuario obtiene información tanto sobre la actividad y la expresión potencial de múltiples enzimas deubiquitinating de una manera rápida y fácil. El método actual es significativamente más eficiente que el uso de anticuerpos individuales para los predichos cien enzimas deubiquitinating en la célula humana.

Células M17 y HeLa cultivadas se recogieron usando el método detallado en la (recolección de células 3.) protocolo y el tejido médula espinal de ratón se obtuvo. El / tejido de la médula espinal sedimento de células se colocó en un tubo con el tampón de lisis descrito en la sección de preparación de reactivos. Los sedimentos celulares se lisaron usando perlas de vidrio (Figura 1A) y muestras de tejido de médula espinal de ratón se homogeneizó usando el homogeneizador (Figura 1B).<...

Watch this Scientific Journal Video about Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples at JoVE.com

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

El actual protocolo detalla un método para medir la actividad de las enzimas funcionalmente homóloga deubiquitinating. Sondas especializadas modifican covalentemente la enzima y permiten la detección. Este método tiene el potencial de identificar nuevas dianas terapéuticas.

Método para medir la actividad de enzimas deubiquitinating en líneas celulares y muestras de tejido

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

method-for-measuring-activity-deubiquitinating-enzymes-cell-lines

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JoVE Journal

Biology

9.5K vistas.  University of Minnesota.  El actual protocolo detalla un método para medir la actividad de las enzimas funcionalmente homóloga deubiquitinating. Sondas especializadas modifican covalentemente la enzima y permiten la detección. Este método tiene el potencial de identificar nuevas dianas terapéuticas. 

Video: Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentación de ensayo, pues el análisis de proteína de unión a la F-actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Investigación

Biología

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

La cuantificación de γH2AX focos en muestras de tejido

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Un ensayo para medir la actividad de Escherichia coli Inducible lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself de dispositivos para la recuperación de las muestras criopreservadas de caída accidental en los tanques de almacenamiento criogénicos

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Medición<em&gt; In Vitro</em&gt; Actividad ATPasa para enzimática Caracterización

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Comparación de tres diferentes métodos para determinar la proliferación celular en líneas celulares de cáncer de mama

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Medición de actividad enzimática de la lactasa en el laboratorio de enseñanza

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Híbrido-Cut: un método de seccionamiento mejorada para muestras de tejido de la planta recalcitrante

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

La disección de la Auditivo Bulla en ratones postnatales: Aislamiento de los huesos del oído medio y el análisis histológico

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-claro: Un tejido claro método de proyección de imagen tridimensional del tejido adiposo

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...