This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

Los procedimientos experimentales con ratones fueron aprobados por el Comité de Cuidado de Animales de la Universidad McGill (protocolo 4966). 1. Preparación de extractos de proteínas de las células cultivadas <ol><li> Lavar las células cultivadas dos veces con 10 ml de fosfato enfriada con hielo de solución salina tamponada (PBS). </li><li> Raspe células adherentes ya sea con una goma de policía o un raspador de células de plástico en 1 ml de suspensión de PBS, la transferencia enfriado con hielo en un tubo de microcentrífuga de 1,5 ml. </li><li> Girar en una microcentrífuga durante 5 min a 700 xg, a 4 ° C. Aspirar PBS. </li><li> Añadir 100 l de tampón de lisis citoplasmática helado (Tabla 1) por 10 7 células, y la pipeta hacia arriba y abajo. </li><li> Incubar en hielo durante 20 min. </li><li> Centrifugado durante 10 minutos a toda velocidad en una microcentrífuga a 4 ° C. </li><li> Pellet Descartar. Transferir el sobrenadante a un nuevo tubo de 1.5 ml y mantener en hielo. </li><li> Detconcentración ermine proteína (normalmente 1 - 10 mg / l) utilizando el ensayo de Bradford 23. </li><li> Alícuotas y celulares tienda extractos a -80 ° C hasta su uso. </li></ol> 2. Preparación de extractos de proteínas de ratón hígado y el bazo <ol><li> La eutanasia a un ratón con inhalación de CO2. </li><li> Coloque el animal sacrificado en un cojín limpio sobre una tabla de disección. Abra el abdomen con unas tijeras. </li><li> Diseccionar el hígado y el bazo mediante el uso de tijeras y fórceps, y enjuagar cada tejido en PBS aproximadamente 50 ml de hielo-frío. </li><li> Inmediatamente cortar tejidos en trozos pequeños con un bisturí (por ejemplo: aproximadamente 1 - 2 mm 3). </li><li> Sin demora, poner trozos de tejidos en un criotubo fresco y luego haga presión para congelar en nitrógeno líquido. Tienda complemento congelados alícuotas de tejido a -80 ° C hasta su uso. </li><li> Homogeneizar una pieza de tejido congelado (aproximadamente 1 - 2 mm 3) en 0,25 hasta 0,5 ml detampón de lisis enfriado con hielo citoplásmica (Tabla 1) con un homogeneizador de tejidos durante 10 s. </li><li> Transferencia homogeneizado a 1,5 ml tubo de microcentrífuga y enfriar en hielo durante 20 min. </li><li> Centrifugado durante 10 minutos a toda velocidad en una microcentrífuga a 4 ° C. </li><li> Deseche pellet y transferencia sobrenadante en un nuevo tubo de microcentrífuga de 1,5 ml. Mantener en hielo. </li><li> Determinar la concentración de proteína (normalmente 1 - 10 mg / l) utilizando el ensayo de Bradford 23. </li><li> Alícuotas y celulares tienda extractos a -80 ° C hasta su uso. </li></ol> 3. Preparación de sonda radiomarcada IRE- <ol><li> Linealizar el plásmido I-12.CAT IRE-que contiene 22 mediante la incubación a 37 ° C durante 1 hora con la endonucleasa de restricción XbaI (1 U por g de plásmido), que escinde aguas abajo de la secuencia IRE. El plásmido linealizado se usó como molde para la transcripción in vitro. </li><li> Configurar unan in vitro reacción de transcripción en un volumen total de 20 l. Utilice las soluciones madre se muestran en la Tabla 2 y añadir: 1 l plantilla plásmido linealizado, tampón de transcripción 4 l; 1 l de mezcla de ATP mezcla / CTP / GTP, 10 l [α- 32 P] -UTP, 2 l de ditiotreitol, inhibidor de RNasa 1 l y 1 l de ARN polimerasa T7. Mezclar pipeteando arriba y abajo. </li><li> Incubar a 40 ° C durante 1 hr 24. </li></ol> 4. Purificación de radiomarcado IRE-sonda <ol><li> Terminar vitro reacción de transcripción in mediante la adición de 1 l de 0,5 M EDTA, pH 8. Mix pipeteando arriba y abajo. </li><li> Añadir 10 l de 10 mg / ml tRNA, como vehículo para una mejor precipitación. Mezclar pipeteando arriba y abajo. </li><li> Añadir 82,5 l de acetato de amonio 3 M. Mezclar por agitación. </li><li> Añadir 273 l de etanol. Mezclar por agitación. </li><li> Dejar reposar a temperatura ambiente durante 5 min. </li><li> Haga girar durante 10 minutos a toda velocidad en una microcentrífuga a RT. Sobrenadante Descartar. </li><li> Lavar el precipitado con 100 l de etanol al 70%. </li><li> Haga girar durante 10 minutos a toda velocidad en una microcentrífuga a RT. Sobrenadante Descartar. </li><li> Aire pellet seco durante 10 min. </li><li> Resuspender pellet en 100 l de doble destilada, autoclave previamente H 2 O. </li><li> Cuantificar la radiactividad en un contador de centelleo líquido, sonda IRE radiomarcado alícuota y almacenar a -80 ° C hasta su uso. Alícuotas congeladas se pueden utilizar durante un máximo de 3 semanas. </li></ol> 5. Preparación de un gel de poliacrilamida nativo para EMSA <ol><li> Montar el gel (16 x 16 cm) mediante el uso de espaciadores 1,5 mm y un peine. </li><li> Para preparar un gel de poliacrilamida nativo 6%, utilice las soluciones de reserva que se muestran en la Tabla 3 Mezclar 7,5 ml de 40% de acrilamida:. Bisacrilamida, 5 ml de 5x TBE y 37,5 mldoble H2O destilada </li><li> Añadir 0,5 ml de persulfato 10% recién preparada de amonio (APS) y 25 l tetrametiletilendiamina (TEMED). </li><li> Inmediatamente vierta la solución de acrilamida al gel y se deja polimerizar. Espere aproximadamente 30 min. </li><li> Montar el aparato de electroforesis con gel, llenar los tanques con TBE 0,5x y conectar con la fuente de alimentación. </li></ol> Ensayo de cambio de movilidad electroforética 6. <ol><li> Diluir 25 g de extracto de proteína a partir de células o tejidos con tampón de lisis citoplásmica (Tabla 1) en un volumen total de 10 l en (concentraciones de proteína más bajas también se pueden utilizar). Mantener en hielo. Si es necesario, añadir 1 l de 1: 4 diluido 2-mercaptoetanol (2-ME) para activar IRP1 latente (concentración final: 2%) 25. </li><li> Diluir la sonda IRE radiomarcado en doble H 2 O destilada a 200.000 cpm / l, desnaturalización por calor a 95 °C durante 1 minuto, y se enfríe a temperatura ambiente durante al menos 5 minutos. </li><li> Configurar una reacción EMSA añadiendo 1 l de sonda IRE radiomarcado al extracto de proteínas. </li><li> Incubar durante 20 min a TA. </li><li> Añadir 1 l de 50 mg / ml de heparina a la reacción (para inhibir las interacciones no específicas de proteínas con la sonda 9) y continuar la incubación durante otros 10 min. Alícuota de la solución madre de heparina (50 mg / ml) y se almacena a -80 ° C. </li><li> Cuando se utilizan sondas radiomarcadas largos (&gt; 60 nucleótidos), añadir 1 l de RNasa T1 (1 U / l) y se incuba durante 10 min a RT para reducir la proteína de unión no específica a la sonda, y para permitir una mejor separación de la ARN / proteína complejo durante la electroforesis. </li><li> Añadir 3 l de tampón de carga (80% de glicerol + azul de bromofenol), mezcla y carga sobre la no desnaturalización gel de poliacrilamida al 6%. </li><li> Ejecutar el gel durante 60 min a 130 V (5 V / cm). </li><li> Transfer el gel sobre un papel de filtro grande y seco. </li><li> Exponer a una película y desarrollar autorradiografía. El tiempo de exposición puede variar de 1 hr (o menos) hasta O / N. </li></ol>

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The authors declare that they have no competing financial interests.

En este documento, se describe un protocolo que se ha desarrollado para estudiar las actividades IRE-unión de IRP1 y IRP2, y mostramos datos representativos. Mediante el uso de diferentes sondas, este protocolo también se puede ajustar para el estudio de otras proteínas de unión al ARN. Un paso crítico es el tamaño de la sonda. El uso de sondas largas, que es común cuando el sitio de unión exacto es desconocido, puede dar lugar a complejos de ARN / proteína que no migran de manera diferente que el ARN libre. En...

La EMSA se desarrolló originalmente para estudiar la asociación de las proteínas de unión al ADN con secuencias de ADN diana 1,2. El principio es similar para las interacciones RNA / proteína 3, que es el foco de este artículo. Brevemente, el ARN está cargado negativamente y migrará hacia el ánodo durante la electroforesis no desnaturalizante de poliacrilamida en (o agarosa) geles. La migración dentro del gel depende del tamaño de la ARN, que es proporcional a su carga. La unión específica de una proteína de ARN altera su movilidad, y los complejos migra más lento en comparación con el RNA libre. Esto se debe principalmente a un aumento en la masa molecular, pero también a alteraciones en la carga y posiblemente conformación. Utilizando un ARN marcado como sonda permite un fácil monitoreo del &quot;retardo en gel&quot; o &quot;bandshift&quot;. El uso de 32 sondas de ARN P-etiquetados es muy común y ofrece una alta sensibilidad. La migración de ARN / complejos de proteínas y ARN libre se detectanpor autorradiografía. Los inconvenientes son la corta vida media de 32 P (14,29 días), el deterioro progresivo de la calidad de la sonda debido a radiólisis, la exigencia de una licencia de radiactividad y la infraestructura para el trabajo de la radiactividad, y posibles problemas de seguridad de la biotecnología. Por lo tanto, los métodos no isotópicos alternativos para marcar la sonda de ARN se han desarrollado, por ejemplo, con fluoróforos o biotina, que permiten la detección mediante formación de imágenes fluorescente o quimioluminiscente 4,5. Las limitaciones de estos métodos son el mayor costo y, a menudo sensibilidad reducida en comparación con marcaje isotópico, y el potencial de marcadores no isotópicos para interferir con la interacción de ARN / proteína. Geles de poliacrilamida no desnaturalizantes son adecuados para la mayoría de aplicaciones EMSA y se usan comúnmente. En ocasiones, geles de agarosa pueden representar una alternativa para el análisis de grandes complejos. La principal ventaja de EMSA es que combina la simplicidad, sensibilidad y robustez 4 &lt;/ Sup&gt;. El ensayo puede completarse en unas pocas horas y no requiere instrumentación sofisticada. Interacciones ARN / proteína pueden ser detectados por la EMSA, en concentraciones tan bajas como 0,1 nM o menos, y dentro de una amplia gama de condiciones de unión (pH 4,0 a 9,5, la concentración de sal monovalente 1 - 300 mM, y la temperatura de 0 - 60 ° C). La formación de complejos de ARN / proteína también puede ser estudiada por el filtro obligatorio de ensayo. Este es un procedimiento simple, rápido y barato basado en la retención de los complejos de ARN / proteína en un filtro de nitrocelulosa, mientras que una sonda de ARN libre pasa a través de 6. En comparación con EMSA, se limita en los casos en que la sonda de ARN contiene múltiples sitios de unión, o el extracto crudo contiene más de una proteínas de unión al ARN que se unen a la sonda en el mismo sitio. Mientras múltiples interacciones RNA / proteína no se detectarán por el filtro obligatorio de ensayo, que se pueden visualizar fácilmente por EMSA. En algunos casos, la visualización es vísperan posible cuando dos complejos de ARN / proteína co-migran (por ejemplo, humano IRP1 / IRE y complejos IRP2 / IRE), mediante la adición de un anticuerpo contra una de las proteínas de unión al ARN a la reacción de EMSA, produciendo más retraso en el gel ( &quot;supershift&quot;) 7. El EMSA se ha utilizado ampliamente para estudiar IRP1 y IRP2, que son reguladores post-transcripcional del metabolismo del hierro 8-10. Operan mediante la unión a IRE, estructuras de horquilla conservadas filogenéticamente dentro de los UTRs de varios mRNAs 11. IREs fueron descubiertos por primera vez en los ARNm que codifican la ferritina y transferrina 12 receptor 1 (TfR1) 13, las proteínas de almacenamiento de hierro y la absorción, respectivamente. Más tarde, IREs se encontraron en erythroid específicos aminolevulinato sintasa (ALAS2) 14, aconitasa mitocondrial 15, ferroportina 16, transportador de metales divalentes 1 (DMT1) 17, factor inducible por hipoxia 2 <em&gt; α (HIF2α) 18, y otra ARNm 19-21. El H- prototipo y ARNm de L-ferritina contienen uno IRE en sus 5 &#39;UTR, mientras TfR1 ARNm contiene múltiples IREs en su extremo 3&#39; UTR. Interacciones IRE / IRP inhiben específicamente ferritina traducción de ARNm bloqueando estéricamente su asociación de los 43S subunidad ribosomal; además, se estabilizan mRNA TfR1 contra la escisión endonucleolítica. IRP1 y compartir IRP2 extensa similitud de secuencia y exhiben alta actividad de unión IRE-en las células hierro de hambre. En las células de hierro repleta, IRP1 reúne un cubane Fe-S clúster que convierte a la aconitasa citosólica a expensas de su actividad de unión a IRE, mientras IRP2 sufre degradación proteasomal. Por lo tanto, las interacciones IRE / IRP dependen del estado del hierro celular, sino que también están regulados por otras señales, tales como H 2 O 2, el óxido nítrico (NO) o hipoxia. Aquí se describe el protocolo para evaluar la actividad de unión a IRE a partir de extractos crudos de células y tejidos por EMSA. Se utilizó una sonda de IRE marcado con 32P H-ferritina que se ha generado mediante transcripción in vitro a partir de una plantilla de ADN plásmido (I-12.CAT), donde la secuencia IRE se introdujo originalmente en orientación sentido aguas abajo del sitio de la polimerasa T7 RNA por clonación de los oligonucleótidos sintéticos hibridados 22.

<table border="0" cellpadding="0" cellspacing="0" width="430">
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		<tr height="17">
			<td height="17" style="height:17px;width:195px;">Name of the Materials&#160;</td>
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			<td height="17" style="height:17px;">leupeptin</td>
			<td>SIGMA</td>
			<td>L2884</td>
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			<td>SIGMA</td>
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			<td height="17" style="height:17px;">BioRad Protein Assay</td>
			<td>BIORAD</td>
			<td>500-0006</td>
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			<td height="17" style="height:17px;">T7 RNA polymerase</td>
			<td>Thermoscientific</td>
			<td>EPO111</td>
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			<td>Beckman Coulter</td>
			<td align="right">141349</td>
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		<tr height="17">
			<td height="17" style="height:17px;">heparin</td>
			<td>SIGMA</td>
			<td>H0777</td>
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			<td height="17" style="height:17px;">Rnase T1</td>
			<td>Thermoscientific</td>
			<td>EN0541</td>
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			<td height="17" style="height:17px;"></td>
			<td></td>
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			<td height="17" style="height:17px;">Name of the Equipments&#160;</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
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			<td height="17" style="height:17px;">Tissue Ruptor</td>
			<td>Qiagen</td>
			<td align="right">9001271</td>
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			<td height="17" style="height:17px;">Scintillation counter</td>
			<td>Beckman Coulter</td>
			<td>LS6500</td>
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			<td height="17" style="height:17px;">Protean II xi Cell&#160;&#160;</td>
			<td>BIORAD</td>
			<td>165-1834</td>
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			<td height="17" style="height:17px;">20 wells combs 1.5mm thick</td>
			<td>BIORAD</td>
			<td>165-1868</td>
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			<td height="17" style="height:17px;">1.5 mm spacers</td>
			<td>BIORAD</td>
			<td>165-1849</td>
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		<tr height="17">
			<td height="17" style="height:17px;">PowerPac</td>
			<td>BIORAD</td>
			<td>164-5070</td>
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electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

Interacciones RNA / proteína son críticos para las vías de regulación post-transcripcional. Entre las proteínas de unión al ARN citosólicos mejor caracterizados son proteínas reguladoras del hierro, IRP1 y IRP2. Se unen al hierro elementos de respuesta (IRES) dentro de las regiones no traducidas (UTRs) de varios mRNAs objetivo, controlando así la traducción o la estabilidad ARNm. Interacciones IRE / IRP han sido ampliamente estudiados por la EMSA. Aquí se describe el protocolo de EMSA para analizar la actividad de unión a IRE de IRP1 y IRP2, que se puede generalizar para evaluar la actividad de otras proteínas de unión al ARN también. Un lisado de proteína cruda que contiene una proteína de unión al ARN, o una preparación purificada de esta proteína, se incuba con un exceso de 32 sonda de ARN marcado con P, lo que permite la formación de complejos. La heparina se añade para impedir la proteína no específica a la unión de la sonda. Posteriormente, la mezcla se analizó por electroforesis no desnaturalizante en gel de poliacrilamida. La sonda libremigra rápido, mientras que el ARN / proteína exposiciones complejas movilidad retrasados; por lo tanto, el procedimiento se denomina también &quot;retardo en gel&quot; o ensayo &quot;bandshift&quot;. Después de la finalización de la electroforesis, el gel se seca y complejos de ARN / proteína, así como sonda libre, se detectan por autorradiografía. El objetivo general del protocolo es detectar y cuantificar IRE / IRP y otras interacciones RNA / proteína. Por otra parte, EMSA también se puede utilizar para determinar la especificidad, afinidad de unión, y la estequiometría de la interacción de ARN / proteína bajo investigación.

Una sonda IRE radiomarcado se prepara, como se describe en las secciones 3 y 4 del protocolo. La secuencia de la sonda era 5&#39;-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUG C UUCAA C AGUGC UUGGA CGGAUCCU-3 &#39;; los nucleótidos en negrita representan un residuo C desapareado y el bucle, que son características críticas de IRE. La radiactividad específica de la sonda fue de 4,5 x 10 9 cpm / g de ARN. Para evaluar los efectos de las perturbaciones de...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Aquí se presenta un protocolo para analizar las interacciones RNA / proteína. El ensayo de cambio de movilidad electroforética (EMSA) se basa en la migración diferencial de los complejos de ARN / proteína y ARN libre durante la electroforesis en gel nativo. Mediante el uso de una sonda de RNA radiomarcada, complejos / proteína de ARN pueden ser visualizados por autorradiografía.

La movilidad electroforética cambio de ensayo (EMSA) para el estudio de las interacciones RNA-proteína: El IRE / IRP Ejemplo

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

53.2K vistas.  Jewish General Hospital.  Aquí se presenta un protocolo para analizar las interacciones RNA / proteína. El ensayo de cambio de movilidad electroforética (EMSA) se basa en la migración diferencial de los complejos de ARN / proteína y ARN libre durante la electroforesis en gel nativo. Mediante el uso de una sonda de RNA radiomarcada, complejos / proteína de ARN pueden ser visualizados por autorradiografía. 

Video: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Investigación

Biología

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Optimizado de gel de fibrina bolas de ensayo para el Estudio de la angiogénesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Los estímulos generados por ordenador Modelo animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

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 ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Una afinidad líquido fase de captura de ensayo utilizando partículas magnéticas para estudiar la interacción proteína-proteína: el ejemplo de poliomyelits-Nanobodies

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

El Protein Complex Plataforma de Producción MultiBac en el EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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

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

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

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

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

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando Manipulación sola molécula y de imagen para el estudio de las interacciones proteína-ADN

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

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

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

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

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

Ensayo de inmunoprecipitación de la cromatina para la Identificación de Arabidopsis proteína-DNA<em&gt; En Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

La detección de funcional no codificante-variantes genéticas Uso de movilidad electroforética Shift ensayo (EMSA) y el ADN afinidad Precipitación Ensayo (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...