Este trabalho foi apoiado por uma comunhão de Ellison/AFAR pós-doutorado no programa de pesquisa de envelhecimento de SJK, e um prêmio da Fundação de Glenn e NIH concede ao PC (1P01AG034906, 1R01GM 090311, 1R01ES 020812). Todos os autores aparecem no filme.

1. tratamento de peptídeos às células <ol><li>placa 2 milhões SH-SY5Y ou células HEK293 (2 x 10 6) em um 10 cm do prato e cultivá-las por 2 dias</li> <li>(opcional) no outro dia, lave as células com Dulbecco isento de soro &#39; s modificada Águia mídia médio (DMEM) uma vez e incubar com soro DMEM livre durante a noite, se o tratamento deve ser feito em meios livres de soro.</li>
	<li>No dia 3, dissolver peptídeos de S14G-humanin em 0,2 µm filtrado, água destilada e reconstituí-los como solução estoque de 1mM. 
	Nota: Os peptídeos devem ser dissolvidos em solvente apropriado, que pode ser determinado pelas características sequência do peptide (por exemplo, carga geral) e pode ser fornecido pelo fabricante se os peptídeos são comercialmente disponíveis. Por exemplo, S14G-humanin, SHLP2 e SHLP6 e MOTS-c se dissolvem na água. Alíquota os peptídeos em um volume adequado (por exemplo, 50 μL) para evitar congelar e descongelar ciclos. Uma vez descongelado, não usar outra vez.</li>
	<li>Alíquota do soro-contendo pré-aquecido ou media serum-free em um Erlenmeyer de 50 mL do tubo e adicionar 1 mM temperatura de solução para torná-lo em uma concentração de trabalho (por exemplo, 1 µM, 10 µM).</li>
	<li>Substituir a mídia com 6 mL de solução de peptídeo da etapa 1.4. e incube as celulas para a quantidade adequada de tempo. 
	Nota: Escolher o tempo de incubação, dependendo de sua condição que ativa ERK.</li>
</ol> 2. Fraccionamento subcelular para o citoplasma e núcleo <ol><li>lavar as células duas vezes com 10 mL de PBS gelado, raspar as células no prato em 5 mL de PBS gelado e transferir a suspensão de células para um tubo cônico de 15 mL.</li>
	<li>Centrifugar as células a 500 x g por 5 min a 4 ° C.</li>
	<li>Aspire a PBS e ressuspender o sedimento em 200 µ l de tampão de fracionamento gelada, (pH HEPES 10mm = 7,6, 3 mM MgCl 2, 10 mM KCl, 5% (v/v) glicerol, 1% não iónico (C 13 H 22 O (C 2 H 4 O) n), inibidores da protease/fosfatase). 
	Nota: Para manter as proteínas no seu estatuto de fosforilação, o inibidor da fosfatase como bem como protease inibidor deve ser adicionado recentemente e as amostras devem ser mantidas no gelo em todos os tempos. Repetição de congelamento e degelo ciclos de amostras devem ser evitados. Amostras de alíquotas em pequena quantidade (por exemplo, 50 μg) antes de congelá-los a -80 ° C.</li>
	<li>Incubar a pelota re-suspensa no gelo por 15 min e em seguida centrifugar a 250 x g por 5 min a 4 ° C.</li>
	<li>Recolher o sobrenadante como a fração citoplasmática e manter a pelota para a fração nuclear.</li>
	<li>De centrifugação o sobrenadante para remover restos celulares e outros contaminantes a 18.000 x g durante 10 minutos a 4 ° C e depois transferir o sobrenadante para um novo tubo de microcentrifugadora. Esta é a fração do citosol.</li>
	<li>Resuspenda o pellet em tampão de lavagem gelada 200 μL (pH HEPES 10mm = 7,6, 1,5 mM de MgCl 2, 420 mM de NaCl, 25% (v/v) glicerol, 0,2 mM EDTA, inibidores da protease/fosfatase) e centrifugar 250 x g por 5 min a 4 ° C</li>
	<li>Remover o sobrenadante e ressuspender o precipitado em 100 μL gelada, nuclear solução tampão (pH HEPES 20 mM = 7,6, 1,5 mM de MgCl 2, 420 mM de NaCl, 25% (v/v) glicerol, 0,2 mM EDTA, inibidores da protease/fosfatase) e proceda à sonicação 10 vezes (5 s, 10 s fora, 30% amp.) na Ice.</li>
	<li>Centrifugar a 18.000 x g durante 10 minutos a 4 ° C.</li>
	<li>Transferir o sobrenadante para um novo tubo de microcentrifugadora. Esta é a fração nuclear.</li>
	<li>Quantify a quantidade de proteína usando um ensaio BCA</li></ol>
 3. fração mitocondrial bruto <ol><li>lavar as células em cada prato de 10 cm com 10 mL de PBS gelado, adicionar 5 mL PBS gelado e separar as células com uma espátula de celular. 
	Nota: Use três pratos de 10cm de células para obter um bom rendimento da mitocôndria para Western Bloting.</li>
	<li>Transferir a suspensão de células para um tubo cônico de 15 mL e combinar tudo a partir de três pratos de 10 cm em um tubo cônico de 15 ml.</li>
	<li>Centrifugar as células a 600 x g durante 10 minutos a 4 ° C.</li>
	<li>Aspire a PBS e ressuspender o sedimento em 1 mL de tampão de isolamento de mitocôndrias gelada (10 mM Tris-MOPS, 1 mM Tris/EGTA, 200 mM de sacarose, ajustar o pH = 7,4).</li>
	<li>Homogeneizar as células com 25 golpes de um homogeneizador de 2 mL com pilão revestida de politetrafluoretileno na Ice. 
	Nota: Este passo é fundamental para manter a integridade mitocondrial e maximizar o rendimento da fração mitocondrial. O número de traços deve ser otimizado para cada tipo de célula. Precool o homogenizador antes de iniciar o procedimento.</li>
	<li>Transferir o homogeneizado para um tubo de microcentrifugadora e centrifugar, a 600 x g durante 10 minutos a 4 ° C para remover núcleos e células ininterrupta.</li>
	<li>Recolher o sobrenadante, transferi-lo para um novo tubo de microcentrifugadora e centrifugá-lo em 7.000 x g durante 10 minutos a 4 ° C. 
	Nota: A pelota é solta, recolher o sobrenadante com cuidado e tente não perturbar a pelota.</li>
	<li>Remover o sobrenadante e ressuspender o sedimento com 200 μL de tampão de isolamento de mitocôndrias gelada, transferir a solução para um tubo de microcentrifugadora novo. Centrifugar o tubo a 7.000 x g durante 10 minutos a 4 ° C.</li>
	Repita o passo 3.8 para lavar o sedimento de <li>. Não é necessário transferir o sobrenadante para um novo tubo de microcentrifuga nesta etapa.</li>
	<li>Retire o pellet lavado o sobrenadante e ressuspender o sedimento contendo mitocôndria com 50 μL de tampão RIPA.</li>
	<li>Incubar a suspensão no gelo durante 10 min.</li>
	<li>Centrifugar a suspensão de 16.000 x g, durante 15 min a 4 ° C.</li>
	<li>Transferir o sobrenadante para um novo tubo de microcentrifugadora. Esta é a fração mitocondrial.</li>
	<li>Quantificar a quantidade de proteína usando um ensaio BCA.</li>
</ol> 4. Mancha ocidental para Phospho-específicas proteínas <ol><li>realizar uma eletroforese em gel de SDS-poliacrilamida (gel de 8-16% premade) e a proteína de transferência para uma membrana PVDF 4.</li>
	<li>Incubar a membrana com 5% BSA em TBST (0,1% polissorbato 20) por 30 min à temperatura ambiente para bloquear sites de fundo inespecificas. 
	Nota: Para a deteção de proteína fosforilada, bloquear a membrana com BSA não leite. O leite contém caseína, um phosphoprotein abundante, o que resulta em alto sinal inespecífico.</li>
	<li>Incubar a membrana com o anticorpo primário (antifosfo-ERK1/2) a 4 ° C durante a noite.</li>
	<li>No dia seguinte, lave a membrana com TBST (0,1% polissorbato 20) três vezes por 5 min à temperatura ambiente.</li>
	<li>Incubar a membrana com anticorpo secundário (anticoelho HRP) por 1h à temperatura ambiente.</li>
	<li>Lavar a membrana com TBST (0,1% polissorbato 20) três vezes por 5 min à temperatura ambiente.</li>
	<li>Incubar a membrana com solução ECL e imagem da membrana no analisador de imagem.</li>
	<li>Tira a membrana com descascamento de buffer para 15 min a temperatura e a lavagem da membrana com TBST três vezes durante 5 min.</li>
	<li>Repita os passos 4.2 a 4.7 usando anticorpo anti-total ERK1/2.</li>
	<li>Para verificar a pureza de cada fração, executar SDS-PAGE e executar immunoblots usando anticorpos reconhecendo marcadores para cada compartimento (por exemplo, B1 jesuina para núcleo, GAPDH para o citosol e TOM20 para as mitocôndrias).</li>
</ol>

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Pinchas Cohen é sócio e consultor para Kelvin CohBar, Inc., Yen atuou como consultor da CohBar, Inc.

Aqui, temos demonstrado que humanin mediada por peptídeo ERK1/2 ativação ocorre em dois diferentes tipos de células, e a Localização subcellular de ERK1/2 registrados pode ser diferente dependendo das condições (por exemplo, dose de peptídeo, ponto do tempo e célula tipo). Tem sido demonstrado que humanin sinais através de dois receptores diferentes22,23, que pode explicar as diferenças na sinalização entre as linhas de duas células, bem c...

Estudos emergentes mostram que peptídeos derivados mitocondriais (MDPs) desempenham importantes funções no metabolismo e cytoprotection1,2,3. Compreensão da via de transdução de sinal na presença de MDPs nos dá insight sobre o mecanismo pelo qual o MDPs modular várias funções. O primeiro PDM identificado, humanin, foi mostrado para aumentar a quinase de sinal-regulado extracellular 1/2 (ERK1/2) fosforilação através de seu receptor ligação4,5. No entanto, o efeito a jusante de ERK1/2 ativação é ainda underexplored.
A cascata de ERK1/2 serve como um mediador essencial em uma variedade de processos celulares, incluindo a proliferação, migração celular, metabolismo celular, sobrevivência e apoptose6,7,8. Para orquestrar todos estes processos celulares distintos, a atividade e Localização subcellular de ERK1/2 estão fortemente regulamentados por fosfatases e proteínas de andaime9,10. Além de modificação pós-traducional, o vaivém dinâmico de ERK1/2 também regula sua sinalização função, atividade e especificidade11,12. ERK1/2 é principalmente localizadas no citosol13. Um conjunto de proteínas de ancoragem e andaime ajudar a reter ERK1/2 elementos do citoesqueleto, na superfície das organelas, ou difusa no citoplasma13. Após a estimulação, ERK1/2 é fosforilada e torna-se dissociado de suas proteínas de ancoragem, permitindo a translocação de ERK1/2 para outros compartimentos subcelulares, incluindo o núcleo, mitocôndrias, Golgi e lisossomos14, 15 , 16.
Embora humanin seja conhecido para ativar a via de sinalização ERK1/2, a ativação de ERK1/2 só é observada no lisado celular total. Conforme descrito anteriormente, desde que a Localização subcellular ERK1/2 desempenha um papel crucial em seu efeito a jusante, a análise de tanto a Localização subcellular e os níveis totais de fosforilada ERK1/2 é necessário para fornecer uma compreensão abrangente de ativação induzida por humanin de ERK1/2 e a ativação dos destinos a jusante.
Para entender as organelas do alvo do ativado ERK1/2, realizou-se o fraccionamento subcelular seguido pela mancha ocidental para fosforilada ERK1/2. Este método pode ser implementado facilmente como ele utiliza reagentes e equipamentos laboratoriais padrão. Os compartimentos subcellular isolados são de alta pureza, permitindo que o resultado deve ser interpretado direta. Immunostaining de ERK1/2 pode produzir resultados semelhantes. No entanto, certos compartimentos subcellular são relativamente difíceis de Visualizar e requerem métodos especiais de fixação e permeabilização. ERK1/2 níveis variam em compartimentos subcelulares, e esta variação pode causar sinais falsos positivos e falsos negativos quando se olha para o lisado celular total. Portanto, um immunoblot usando compartimentos subcellular isolados dá-em uma melhor compreensão da localização ERK1/2.
A versatilidade do método permite modificações do protocolo para investigar os efeitos de outros estimulantes, incluindo outros MDPs ou translocação de outras moléculas de sinalização como STAT3. Recentemente descobertos pequenos humanin, como peptídeos (corsários) são codificados da região de rRNA 16S onde humanin é codificado, e eles têm propriedades semelhantes mas distintas em relação ao HN17. Por exemplo, SHLP2 e SHLP3 ativam ERK1/2 após 8 h embora humanin ativa ERK1/2 dentro de 5 min. Localização subcellular exame de ERK1/2, em resposta a diferentes peptídeos nos dará uma melhor compreensão da biologia destes peptides. Emergentes evidências mostraram que a Localização subcellular das moléculas de sinalização desempenha um papel crucial em seus efeitos a jusante. Por exemplo, STAT3 é tradicionalmente conhecida para ser localizada principalmente no citosol em células de descanso, e então ele translocates até ao núcleo para ativar a expressão gênica em resposta a citocinas18. STAT3 também translocates à mitocôndria e regula o ciclo TCA e de produção de ATP19. Sobre regulamento de autofagia, diferente Localização subcellular de STAT3 modula autofagia em várias maneiras20. Por exemplo, nuclear STAT3 transcricionalmente regula genes relacionados autofagia e atua como um modulador de autofagia. Citoplasmáticos STAT3 constitutivamente inibe a autofagia interagindo com moléculas de sinalização de autofagia. Mitocondrial STAT3 inibe e impede mitophagy suprimindo o estresse oxidativo induzido por autofagia. Portanto, esse método de isolamento do compartimento subcellular é crucial para compreender o papel da outra sinalização moléculas bem 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

Peptídeos derivados mitocondrial (MDPs) são uma nova classe de peptídeos que são codificados por frames de leitura abertos pequenos dentro de outros genes conhecidos do genoma mitocondrial. MDPs têm uma grande variedade de efeitos biológicos como protegendo os neurônios da apoptose, melhorando marcadores metabólicos e protegendo as células da quimioterapia. Humanin foi o MDP primeiro a ser descoberto e o peptídeo mais estudado entre a família MDP. Os receptores de membrana e vias de sinalização a jusante de humanin foram cuidadosamente caracterizadas. MDPs adicionais tais como MOTS-c e SHLP1-6 foram descobertos mais recentemente e os mecanismos de sinalização ainda precisa ser elucidado. Aqui nós descrevemos um método de cultura com base de célula para determinar a função destes peptides. Em particular, técnicas de fracionamento celular em combinação com mancha ocidental permitem a determinação quantitativa da ativação e translocação de importante molécula de sinalização. Embora existam outros métodos de fracionamento celular, o descrito aqui é um método fácil e simples. Estes métodos podem ser usados para elucidar ainda mais o mecanismo de ação desses peptídeos e outros agentes terapêuticos.

Usando o procedimento apresentado aqui, tratamos de células HEK293 e SH-SY5Y com 1 μM e 100 μM S14G-humanin, um potente humanin analógico21, respectivamente, em completa mídia para os períodos de tempo indicado (Figura 1A e Figura 1 B). em seguida examinamos a forma fosforilada e total de ERK1/2 em Thr202/Tyr204 de extratos de proteínas totais...

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 descreve como estimulam as células com peptídeos derivados mitocondriais e avaliar a cascata de sinalização e localização de fosfo-proteínas.

Fraccionamento subcelular para activação de ERK tratamento de peptídeo derivado mitocondrial

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 visualizações.  University of Southern California. Este protocolo descreve como estimulam as células com peptídeos derivados mitocondriais e avaliar a cascata de sinalização e localização de fosfo-proteínas.

Vídeo: 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.

Preparação e fracionamento de Xenopus laevis Egg Extractos

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

Biologia

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.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery 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.

Fracionamento em Subcelulares situ de aderentes e não aderentes células de mamíferos

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 Assay Activation GTPase

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.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

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.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

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 Estruturas de imagem Subcelulares em ratos vivos Expressando fluorescentes Proteínas

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.

Imagens de células Lesão de Membrana e Processos Subcelulares envolvidas no reparo

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 avaliar Subcelulares Compartimentos de músculo em<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).