Este trabajo fue financiado por una Beca Postdoctoral de Ellison/lejos en el programa de investigación envejecimiento a Santa Justa Klan, y un premio de la Fundación de Glenn y NIH concede a PC (1P01AG034906, 1R01GM 090311, 1R01ES 020812). Todos los autores aparecen en la película.

1. tratamiento de péptidos a las células <ol><li>placa 2 millones SH-SY5Y o células HEK293 (2 x 10 6) en 10 cm plato y cultivan durante 2 días</li> <li>(opcional) el siguiente día, lavar las células con el suero Dulbecco &#39; s modificado Águila media media (DMEM) una vez y se incuba con suero DMEM libre toda la noche si el tratamiento debe hacerse en medios libres de suero.</li>
	<li>El día 3, disolver humanin S14G péptidos en 0.2 μm filtrada, agua destilada y reconstituir como solución madre de 1mM. 
	Nota: Los péptidos deben ser disuelto en el disolvente apropiado, que puede ser determinado por las características de la secuencia del péptido (p. ej., carga general) y puede ser proporcionado por el fabricante si los péptidos están comercialmente disponibles. Por ejemplo, humanin S14G, SHLP2 y SHLP6 y MOTS-c se disuelven en agua. Alícuota los péptidos en un volumen adecuado (por ejemplo, 50 μL) para evitar congelar y descongelar los ciclos. Una vez descongelado, no usarlos otra vez.</li>
	<li>Alícuota el precalentado del suero que contienen o medio libre de suero en un cónico de 50 mL tubo y temperatura solución stock de 1 mM para convertirla en una concentración del trabajo (p. ej., 1 μm, 10 μm).</li>
	<li>Reemplazar los medios de comunicación con 6 mL de solución de péptido de paso 1.4. e incube las células por la cantidad de tiempo apropiada. 
	Nota: Elija el tiempo de incubación dependiendo de la condición que activa ERK.</li>
</ol> 2. Fraccionamiento subcelular de citosol y núcleo <ol><li>lavan las células dos veces con 10 mL de PBS helado, raspar las células de la placa en 5 mL de PBS helado y transferir la suspensión de células en un tubo cónico de 15 mL.</li>
	<li>Centrifugar las células a 500 x g durante 5 min a 4 ° C.</li>
	<li>Aspirar el PBS y resuspender el pellet en 200 μL de tampón de fraccionamiento helada, (pH HEPES 10 mM = 7.6, 3 mM MgCl 2, 10 mM KCl, 5% (v/v) de glicerol, 1% de tensioactivo no iónico (C 13 H 22 O (C 2 H 4 O) n), inhibidores de la proteasa/fosfatasa). 
	Nota: Para mantener las proteínas en su estado de fosforilación, el inhibidor de la fosfatasa como bien como proteasa inhibidor debe añadirse recientemente y las muestras deben conservarse en hielo en todo momento. Repetición de congelación y deshielo deben evitarse los ciclos de las muestras. Muestras alícuotas en pequeña cantidad (por ejemplo 50 μg) antes de congelar a -80 ° C.</li>
	<li>Incubar el sedimento resuspendido en hielo durante 15 min y luego centrifugar a 250 x g durante 5 min a 4 ° C.</li>
	<li>Recoger el sobrenadante como la fracción citoplasmática y mantener el sedimento de la fracción nuclear.</li>
	<li>Centrifugar el sobrenadante para eliminar los residuos celulares y otros contaminantes a 18.000 x g por 10 min a 4 ° C y luego transferir el sobrenadante a un tubo nuevo de microcentrífuga. Se trata de la fracción citosol.</li>
	<li>Resuspender el precipitado en tampón de lavado helada de 200 μL (pH HEPES 10 mM = 7.6, 1,5 mM de MgCl 2, 420 mM NaCl, 25% (v/v) de glicerol, 0,2 mM EDTA, inhibidores de la proteasa/fosfatasa) y centrifugar a 250 x g durante 5 min a 4 ° C</li>
	<li>Quite el sobrenadante y resuspender el precipitado en tampón de extracción helada nuclear de 100 μL (pH HEPES 20 mM = 7.6, 1,5 mM de MgCl 2, 420 mM NaCl, 25% (v/v) de glicerol, 0,2 mM EDTA, inhibidores de la proteasa/fosfatasa) y someter a ultrasonidos 10 veces (5 s a 10 s descuento 30% amp.) en ICE</li>
	<li>Centrifugar a 18.000 x g por 10 min a 4 ° C.</li>
	<li>Transferir el sobrenadante a un tubo nuevo de microcentrífuga. Se trata de la fracción nuclear.</li>
	<li>Cuantificar la cantidad de proteína usando un análisis BCA</li></ol>
 3. fracción mitocondrial cruda <ol><li>Lave las células en cada plato 10 cm con 10 mL de PBS helado, añadir 5 mL PBS helado y separar las células usando un raspador celular. 
	Nota: Usar tres platos de 10 cm de las células para obtener un buen rendimiento de mitocondrias para Western Blotting.</li>
	<li>Transferir la suspensión de células a un tubo cónico de 15 mL y combinar de tres platos de 10 cm en un tubo cónico de 15 ml.</li>
	<li>Centrifugar las células a 600 x g por 10 min a 4 ° C.</li>
	<li>Aspirar el PBS y vuelva a suspender el sedimento en 1 mL de tampón de aislamiento de mitocondrias helada (10 mM Tris-MOPS, 1 mM Tris/EGTA, 200 mM de sacarosa, ajustar a pH = 7.4).</li>
	<li>Homogeneizar las células con 25 golpes de un homogeneizador de 2 mL con Maja recubierto de politetrafluoroetileno en hielo. 
	Nota: Este paso es vital para mantener la integridad mitocondrial y maximizar el rendimiento de la fracción mitocondrial. El número de trazos debe ser optimizado para cada tipo de célula. Preenfriar el homogenizador antes de comenzar el proceso.</li>
	<li>Transferir el homogenizado a un tubo de microcentrífuga y centrifugar a 600 x g por 10 min a 4 ° C para eliminar los núcleos y células intactas.</li>
	<li>Recoger el sobrenadante, transferir a un tubo nuevo de microcentrífuga y centrifugue a 7.000 x g durante 10 min a 4 ° C. 
	Nota: El pellet es flojo, recoger el sobrenadante con cuidado y trate de no perturbar el sedimento.</li>
	<li>Quite el sobrenadante, Resuspender el pellet con 200 μL de tampón de aislamiento de mitocondrias helada y transferir la solución a un tubo nuevo de microcentrífuga. Centrifugar el tubo a 7.000 x g por 10 min a 4 ° C.</li>
	Repita el paso 3.8 para lavar el sedimento <li>. No es necesario transferir el sobrenadante a un tubo nuevo de microcentrífuga en este paso.</li>
	<li>Retire el sobrenadante de la pelotilla de lavado y vuelva a suspender el sedimento que contiene mitocondrias con 50 μL de tampón RIPA.</li>
	<li>Incubar la suspensión en hielo durante 10 minutos</li>
	<li>Centrifugar la suspensión a 16.000 x g durante 15 min a 4 ° C.</li>
	<li>Transferir el sobrenadante a un tubo nuevo de microcentrífuga. Se trata de la fracción mitocondrial.</li>
	<li>Cuantificar la cantidad de proteína usando un ensayo BCA.</li>
</ol> 4. Western blot para proteínas fosfo-específicos <ol><li>realizar una electroforesis del gel de SDS-poliacrilamida (8-16% preconfeccionado gel) y la proteína de la transferencia a una membrana PVDF 4.</li>
	<li>Incubar la membrana con 5% de BSA en TBST (0.1% polisorbato 20) por 30 min a temperatura ambiente para bloquear sitios de Unión inespecífica de fondo. 
	Nota: Para la detección de la proteína fosforilada, bloquear la membrana con BSA no leche. La leche contiene caseína, una fosfoproteína abundante, que se traduce en señal inespecífica de alta.</li>
	<li>Incubar la membrana con el anticuerpo primario (anti-fosfo-ERK1/2) a 4 ° C durante la noche.</li>
	<li>Al día siguiente, lavar la membrana con TBST (0.1% polisorbato 20) tres veces durante 5 min a temperatura ambiente.</li>
	<li>Incubar la membrana con el anticuerpo secundario (anti-conejo HRP) por 1 h a temperatura ambiente.</li>
	<li>Lavar la membrana con TBST (0.1% polisorbato 20) tres veces durante 5 min a temperatura ambiente.</li>
	<li>Incubar la membrana con solución de ECL y la membrana en el analizador de imagen de la imagen.</li>
	<li>Tira de la membrana con stripping buffer durante 15 min a temperatura ambiente y lavar la membrana con TBST tres veces durante 5 minutos</li>
	<li>Repita los pasos del 4.2 al 4.7 usando el anticuerpo de anti-total ERK1/2.</li>
	<li>Para comprobar la pureza de cada fracción, correr de SDS-PAGE y realizar immunoblots utilizando anticuerpos reconocer marcadores para cada compartimiento (p. ej., Lamin B1 núcleo, GAPDH de citosol y TOM20 de mitocondrias).</li>
</ol>

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Pinchas Cohen es un accionista y consultor de CohBar, Inc. Kelvin Yen ha servido como consultor para CohBar, Inc.

Aquí, demostramos que humanin mediada por el péptido de la activación de ERK1/2 se presenta en dos diferentes tipos de células, y la localización subcelular de activados ERK1/2 puede ser diferente dependiendo de las condiciones (p. ej., dosis de péptido, punto del tiempo y de la célula tipo). Se ha demostrado que humanin señales a través de dos diversos receptores22,23, que puede explicar las diferencias en la señalización entre las dos varied...

Emergentes los estudios demuestran que los péptidos derivados de mitocondrial (MDPs) desempeñan papeles importantes en citoprotección y metabolismo1,2,3. Comprensión de la vía de transducción de señal en presencia de MDPs nos da información sobre el mecanismo por el cual MDPs modulan varias funciones. El primer identificado MDP, humanin, se ha demostrado para aumentar la quinasa regulada por señal extracelular 1/2 (ERK1/2) fosforilación a través de su receptor binding4,5. Sin embargo, el efecto aguas abajo de la activación de ERK1/2 es todavía underexplored.
La cascada de ERK1/2 sirve como un mediador esencial en una variedad de procesos celulares como proliferación, migración de la célula, metabolismo celular, supervivencia y apoptosis6,7,8. Para organizar todos estos distintos procesos celulares, la actividad y la localización subcelular de ERK1/2 están estrictamente regulados por fosfatasas y proteínas andamio9,10. Además de la modificación poste-de translación, el transporte dinámico de ERK1/2 regula también su señalización función, actividad y especificidad11,12. ERK1/2 se localiza principalmente en el citosol13. Un conjunto de proteínas de anclaje y andamio ayudar a retener ERK1/2 en elementos del citoesqueleto, sobre la superficie de los organelos, o difuso en el citoplasma13. Sobre el estímulo, ERK1/2 es fosforilada y se convierte en disociarse de sus proteínas de anclajes, permitiendo la translocación de ERK1/2 en otros compartimientos subcelulares, incluyendo el núcleo, mitocondrias, Golgi y lisosomas14, 15 , 16.
Aunque humanin es conocido por activar la vía de señalización de ERK1/2, la activación de ERK1/2 sólo se observa en los lysates de la célula total. Como se describió anteriormente, ya que la localización subcelular de ERK1/2 desempeña un papel crucial en su efecto aguas abajo, el análisis de la localización subcelular y los niveles totales de ERK1/2 fosforilado es necesario para proporcionar una comprensión global de humanin inducida por activación de ERK1/2 y la activación de objetivos posteriores.
Para entender los organelos objetivo activados ERK1/2, se realizó el fraccionamiento subcelular seguida de western blot para fosforilada ERK1/2. Este método se puede implementar fácilmente ya que utiliza reactivos y equipo normal de laboratorio. Los compartimientos subcelulares aislados son de alta pureza, permitiendo que los resultados de interpretarse directamente. Inmunotinción de ERK1/2 puede producir resultados similares. Sin embargo, ciertos compartimientos subcelulares son relativamente difíciles de visualizar y requieren métodos especiales de fijación y permeabilización. ERK1/2 niveles varían en compartimientos subcelulares, y esta variación podría causar señales falsas positivas y falsas negativas en lisados de células enteras. Por lo tanto, un immunoblot usando compartimientos subcelulares aislados nos da una mejor comprensión de la localización de ERK1/2.
La versatilidad del método permite modificaciones del Protocolo a investigar los efectos de otros estimulantes, incluyendo otros MDPs o translocación de otras moléculas de señalización como STAT3. Recientemente descubiertos pequeños péptidos humanin-como (SHLPs) se codifican de región de rRNA 16S donde humanin está codificada, y tienen propiedades similares pero distintos en comparación con HN17. Por ejemplo, SHLP2 y SHLP3 activarán ERK1/2 después de 8 h aunque humanin activa ERK1/2 en 5 minutos examen localización subcelular de ERK1/2 en respuesta a péptidos diferentes nos dará una mejor comprensión de la biología de estos péptidos. Nuevas pruebas demostraron que la localización subcelular de las moléculas de señalización juega un papel crucial en sus efectos aguas abajo. Por ejemplo, STAT3 es conocido tradicionalmente a localizarse principalmente en el citosol de las células de reclinación, y luego transloca en el núcleo para activar la expresión génica en respuesta a citoquinas18. STAT3 también se transloca a la mitocondria y regula el ciclo de TCA y ATP producción19. Con respecto a la regulación de la autofagia, diferente localización subcelular de STAT3 modula la autofagia en varias maneras20. Por ejemplo, STAT3 nuclear transcripcionalmente regula genes relacionados con la autofagia y actúa como un modulador de la autofagia. STAT3 citoplásmico constitutivamente inhibe la autofagia interactuando con moléculas señalizadoras de autofagia. STAT3 mitocondrial inhibe y previene la mitofagia suprimiendo el estrés oxidativo inducido por autofagia. Por lo tanto, este método de aislamiento del compartimento subcelular es crucial para entender el papel de otras moléculas de señalización así como ERK1/2.

<table>
	<tbody>
		<tr>
			<td>p44/42 MAPK (ERK1/2)</td>
			<td>Cell signaling</td>
			<td>9102</td>
			<td>Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)</td>
			<td>Cell signaling</td>
			<td>4370</td>
			<td>Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>Lamin B1</td>
			<td>Cell signaling</td>
			<td>12586</td>
			<td>Nuclear Marker, Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>Cell signaling</td>
			<td>5174</td>
			<td>Cytoplasmic marker, Dilution 1:2,000</td>
		</tr>
		<tr>
			<td>Tom20</td>
			<td>Santa cruz</td>
			<td>SC-17764</td>
			<td>Mitochondria marker, Dilution 1:2,000</td>
		</tr>
		<tr>
			<td>anti-Rabbit-HRP conjugated</td>
			<td>Cell signaling</td>
			<td>7074</td>
			<td>Dilution 1:30,000</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Culture Dish</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>12556002</td>
			<td></td>
		</tr>
		<tr>
			<td>HNG peptide</td>
			<td>Genescript</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25mm sylinge filter</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>09-719A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>3609</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HCL</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>FALCON</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt&#8482; Protease and Phosphatase Inhibitor Cocktail (100X)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>78440</td>
			<td></td>
		</tr>
		<tr>
			<td>Thomas Pestle Tissue Grinder Assemblies with Smooth Pestles</td>
			<td>Thomas Scientific</td>
			<td>3432S90</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>8-16% Mini-PROTEAN TGX Precast Protein Gels</td>
			<td>BIO RAD</td>
			<td>4561104</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Module</td>
			<td>BIO RAD</td>
			<td>1658030</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>BIO RAD</td>
			<td>1704150</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td>
			<td>BIO RAD</td>
			<td>1704272</td>
			<td></td>
		</tr>
		<tr>
			<td>Clarity Western ECL Blotting Substrates</td>
			<td>BIO RAD</td>
			<td>1705060</td>
			<td></td>
		</tr>
		<tr>
			<td>Restore Western blot stripping buffer</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>21059</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator, Medel: FB120</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>695320-07-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment

Péptidos de origen mitocondrial (MDPs) son una nueva clase de péptidos que son codificadas por Marcos de lectura abiertos pequeños dentro de otros genes conocidos del genoma mitocondrial. MDPs con una amplia variedad de efectos biológicos tales como proteger a las neuronas de la apoptosis, la mejora de marcadores metabólicos y protegiendo las células de la quimioterapia. Humanin fue el primer MDP por descubrir y es el péptido más estudiado entre la familia MDP. Los receptores de membrana y vías de señalización corriente abajo de humanin se han caracterizado cuidadosamente. MDPs adicionales como MOTS-c y SHLP1-6 han sido descubiertos más recientemente y los mecanismos de señalización tienen todavía ser aclarada. Aquí describimos un método basado en la cultura de célula para determinar la función de estos péptidos. En particular, técnicas de fraccionamiento celular en combinación con western blot permiten la determinación cuantitativa de la activación y translocación de la molécula de señalización importante. Mientras que hay otros métodos de fraccionamiento celular, descrito aquí es un método fácil y sencillo. Estos métodos pueden utilizarse para aclarar más lejos el mecanismo de acción de estos péptidos y otros agentes terapéuticos.

Utilizando el procedimiento presentado aquí, tratamos las células HEK293 y SH-SY5Y con 1 μM y 100 μM S14G-humanin, un análogo humanin potente21, respectivamente, en completar los medios de comunicación durante los períodos de tiempo indicado (figura 1 y figura 1 B). a continuación examinamos la forma fosforilada y total de ERK1/2 en Thr202/Tyr204</...

Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Este protocolo describe cómo estimular las células con péptidos derivados de mitocondrial y evaluar la cascada de señalización y localización de fosfo-proteínas.

Fraccionamiento subcelular para la activación de ERK a péptidos derivados de mitocondrial tratamiento

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

subcellular-fractionation-for-erk-activation-upon-mitochondrial

Research

JoVE Journal

Biology

7.8K vistas.  University of Southern California. Este protocolo describe cómo estimular las células con péptidos derivados de mitocondrial y evaluar la cascada de señalización y localización de fosfo-proteínas.

Video: Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Preparación y fraccionamiento de los extractos de huevo Xenopus laevis

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Investigación

Biología

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

En Fraccionamiento subcelular situ de células mamíferas adherentes y no adherentes-

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

RHOC GTPasa ensayo de activación

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabólicas etiquetado y de membrana de fraccionamiento para Comparativo Proteómica Análisis de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensión celular

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopia intravital para formar imágenes de estructuras subcelulares en ratones vivos que expresan proteínas fluorescentes

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

Imágenes de células lesión de la membrana y procesos subcelulares implicadas en la reparación

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Métodos para evaluar subcelulares compartimentos de músculo en<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the spotlight and has been shown to have multiple major functions like actively modifying the extracellular matrix and forming an endocrine organ with the lacunocanalicular system that encloses it sending messages to distant sites. Owing to the methods that made it possible to test the osteocyte in vitro from isolating primary osteocytes to osteocyte-like cell lines, osteocytes are now experiencing a resounding interest and a surge of knowledge on structure and function. Many aspects of the osteocyte biology and interaction with other molecular components are yet to be discovered. In this protocol, we describe in detail the efficient isolation of primary osteocytes from dmp1-topaz neonatal mouse calvaria, which express the green fluorescent protein in osteocytes, through cell fractionation and subsequently acquiring cultures of primary osteocytes by FACS.

This protocol describes the dissection of neonatal dmp1-topaz mouse calvaria and isolation of osteocytes expressing the green fluorescent protein through cell digestion and fractionation, in addition to osteocyte preparation for fluorescence activated cell sorting (FACS).