This work was supported by FIRB-Merit grants RBNE08YFN3_005 and RBNE08HWLZ_012, and the Italian Ministry of Economy and Finance to the CNR for the Project &#8220;FaReBio di Qualit&#224;&#8221;.

1. Prepare soluções siRNA <ol><li> Ressuspender 5 nmol de embaralhada (Ctrl) e 5 nmol de BRCA2 siRNA peletes secos em 100 ul de água isenta de RNase (concentração final de siRNA: 50 uM). Este é o banco de siRNA e deve ser armazenado a -20 ° C em alíquotas de 10 ul. </li><li> Tomar uma aliquota de 10 ul de estoque de siRNA e adicionar 40 ul de água isenta de RNase para obter a solução de trabalho a 10 ^ M de siARN. Esta diluição deve ser armazenado a -20 ° C até à sua utilização. NOTA: A solução de trabalho siRNA não devem ser submetidos a congelamento / descongelamento mais de 3 vezes. </li></ol> 2. Sementeira de linhas celulares epiteliais humanas <ol><li> Remover o meio de crescimento de linhas celulares epiteliais humanas que crescem como monocamadas celulares em placas de cultura de tecidos numa incubadora humidificada a 37 ° C, 5% de CO 2. </li><li> Lavam-se as células na placa uma vez com PBS temperatura ambiente (10 ml de PBS para uma placa de 100 mm). </li><li> Para retirar as células a partir da placa,adicionar 2 ml de tripsina a 0,25% / solução de 0,53 mM de EDTA e incubar 3-5 minutos, dependendo do tipo de célula. </li><li> Antes de recolher as células destacadas, neutralizar a tripsina por adição de 2 ml de meio de crescimento completo contendo 10% de soro fetal bovino. </li><li> Transferir as células para um tubo de 15 ml. </li><li> Centrifugar as células a 320-330 x g durante 3 min a 4 ° C num rotor de balde oscilante. </li><li> Ressuspender as peletes de células em 5 ml de meio livre de antibióticos. </li><li> Contar as células ao microscópio numa câmara de Burker, quer utilizando um sistema automatizado de contagem de acordo com as instruções do fabricante. </li><li> Semente as células em placas de 6 poços para ser cerca de 80% confluente após 24 horas (0,5-1 x 10 6 células em 2 ml de meio para cada poço). O número de células óptimas a serem semeadas podem variar dependendo das linhas de células específicas e a taxa de crescimento. NOTA: Não adicionar antibióticos a médio neste momento porque os antibióticos irá interferir negativamente com a eficiência de transfection. É muito importante que as células estão na cultura de passagens baixas (&lt;10) para atingir uma elevada eficiência de transfecção siRNA BRCA2. </li></ol> 3. Células de transfecção com siRNA <ol><li> Vinte e quatro horas após o plaqueamento das células, prosseguir com a primeira rodada de transfecção. <ol><li> Dilui-se 6 ul de 10 siRNA reagente de transfecção à base de lípidos específicos, em 130 ul de meio de soro reduzido. </li><li> Dilui-se 3 ul de siRNA 10 uM (30 pmol) em 130 ul de meio de soro reduzido. </li><li> Combinar o siARN diluiu-se com o reagente de transfecção diluído e incubar durante 5-10 min à temperatura ambiente. </li><li> Durante a incubação, remover o meio das células e adicionar 2 ml de meio fresco pré-aquecido a 37 ° C (sem antibióticos). </li><li> Adicionar 250 ul de complexos de siRNA-reagentes de transfecção para as células (este valor é para um único poço de uma placa de 6 poços). </li><li> Incubam-se as células numa incubadora humidificada a 3776; C com 5% de CO 2. </li></ol></li><li> Após 24 horas, prosseguir com a segunda rodada de transfecção por passos 3.1.1-3.1.5 repetindo com a seguinte modificação para a etapa 3.1.2: diluir 5 ul de 10 mM siRNA (50 pmol) em 130 mL de meio de soro reduzido. <ol><li> Incubam-se as células durante 1-2 dias a 37 ° C antes da análise. NOTA: Duas rodadas de transfecções e uso de uma relação de reagente alta siRNA / transfecção na segunda rodada são essenciais para obter alta eficiência do BRCA2 silenciamento. </li></ol></li></ol> 4. Lisar as células para análise de imunotransferência <ol><li> Preparar tampão de lise fresco com PBS (pH 7,4) contendo 1% do não iónico, não-detergente desnaturante octilfenoxipolietoxietanol, pirofosfato de sódio a 5 mM, ortovanadato de sódio 1 mM, fluoreto de sódio 10 mM, fluoreto de fenilmetilsulfonilo 2 mM, 10 ug / ml de leupeptina, 10 ug / ml de aprotinina. Mantê-lo em gelo. </li><li> Remover o meio das células e wash-los uma vez com PBS frio (2 ml / poço) na placa. Para evitar qualquer transição PBS, removê-la completamente a partir da placa. </li><li> Adicionar 200 ul de tampão de lise a cada poço. NOTA: Se as células têm de ser usado também para outros ensaios, trypsinize as células tal como descrito no passo 2,1-2,6 e ressuspender as células em tampão / meio apropriado de acordo com a aplicação a ser utilizada. </li><li> Rodar suavemente a placa para distribuir uniformemente o tampão de lise e incubar durante 15 min a 4 ° C num rotor. </li><li> Recolhe-se o lisado celular num tubo de microcentrífuga em gelo usando um raspador de células. </li><li> Centrifugar a 17.900 xg durante 25 min a 4 ° C e recolher o sobrenadante num novo tubo. </li><li> Medir a concentração de proteína usando um ensaio de proteína disponível comercialmente de acordo com as instruções do fabricante. </li></ol> Análise immunoblotting 5. BRCA2 <ol><li> Preparar o tampão de corrida, diluindo 50 ml de 20x de Tris-Acetato de SDS execução bpode ser prejudicado (Tricina 50 mM, 50 mM Tris Base, 0,1% de SDS, pH 8,24) com 950 ml de água destilada. </li><li> Preparar a amostra como se segue: adicionar 15? G de lisado celular total, 5 uL de tampão de amostra 4x contendo sulfato de dodecilo de lítio, a pH 8,4, 2 mL de amostra de 10x agente redutor (DTT 0,5 M) e tampão de lise para um volume final de 20 ul. NOTA: Este protocolo permite a detecção de BRCA2 em linhas de células normais, também quando se utiliza uma menor quantidade de proteínas totais (inferior a 5 ug). </li><li> Desnaturar amostras a 55 ° C durante 10 min. NOTA: É fundamental usar 55 ° C. Desde que a proteína BRCA2 é termossensível 11, as temperaturas de desnaturação mais elevadas resultam em perda consistente dos intacto de comprimento completo (390 kDa) da proteína BRCA2. </li><li> Defina-se a câmara de electroforese de acordo com as instruções do fabricante. </li><li> Coloque as amostras e 10 ul de alto peso molecular pré-corados padrão de proteína em um gel precasted (Tris-Acetato de 3-8%) e executado em 120 V para aba 2 horas 12. Parar a corrida eletroforética antes do 55 kDa marcador azul deixa o gel precasted. </li><li> Preparar o tampão de transferência com 50 ml de tampão de transferência 20x, 100 ml de metanol a 100% e 850 ml de água. </li><li> Activar a membrana de PVDF (tamanho do poro de 0,45 um) por imersão em 100% de metanol durante 5 minutos, enxaguar com água destilada e equilibrar em tampão de transferência, pelo menos, durante 5 min. Soak as esponjas do aparelho de transferência com tampão de transferência a 4 ° C até à sua utilização. </li><li> Quando a corrida eletroforética é longo, montar o sanduíche na pilha de transferência na seguinte ordem (bottom-up): três esponjas saturadas em tampão de transferência, uma folha de papel 3MM grau saturado em tampão de transferência, o gel, membrana PVDF ativado, um 3MM folha de papel grau saturado em tampão de transferência, três esponjas saturado em tampão de transferência 13,14. Colocar a pilha no aparelho e as proteínas de transferência em 350 mA durante 4 horas a 4 ° C ou a 180 mA durante a noite a 4 ° C. NOTA: Ser BRCA2 uma proteína muito grande, é crucial para estender o tempo de transferência de 1 hora (tal como sugerido pelo fabricante, e a maioria dos protocolos de transferência) a 4 horas ou durante a noite para assegurar a transferência completa de proteínas de alto peso molecular. Além disso, devido ao longo tempo de transferência, que é essencial para realizar o blotting numa sala fria a 4 ° C para minimizar o sobreaquecimento e facilitar a desmontagem sanduíche. Sistemas de transferência semi-secas não são bons para a transferência de grandes proteínas. </li><li> Após a transferência, lavar a membrana com TBS, bloqueá-lo durante 1 hora à temperatura ambiente em TBS-Tween 0,25% (TBS-T) contendo 5% de leite seco sem gordura e a sonda durante a noite a 4 ° C com 30 ul de anti-anticorpo policlonal de coelho BRCA2 ( 200 ug / ml), diluído em 9 ml de TBS-T + 5% de leite em pó magro [1: 300 (v / v)] de diluição. </li><li> Lava-se a membrana três vezes (10 minutos cada) com TBS-T, em seguida, o filtro incubar durante 1 hora com 1 ml de peroxidase de rábano conjugado com anti-anticorpo secundário de coelho diluído em 10 ml de TBS-T + 5% de leite em pó magro. Em seguida, lava-se a membrana três vezes (10 minutos cada) com TBS-T. </li><li> Realizar imunodetecção por ECL para revelar proteína BRCA2 usando quimioluminescência aumentada (ECL) de Reagente de Detecção de Western Blotting, seguindo as instruções do fabricante. Visualize bandas imunoquimioluminométricos sobre filmes de alta resolução ECL. </li><li> Realizar imunodetecção com um anticorpo para um gene de manutenção, como β-tubulina (55 kDa), no mesmo filtro como controlo de carga, utilizando um anticorpo monoclonal para β-tubulina diluído a 1: 1000 (10 ul de anticorpo anti-tubulina em 10 ml de TBS-T + 5% de leite seco não gordo) durante 1 h à TA. Lavar e incubar o anticorpo secundário como descrito para BRCA2 (5,10-5,11), excepto na utilização de peroxidase de rábano conjugado com anti-ratinho em vez do anticorpo secundário anti-coelho. </li><li> Adquirir uma imagem digital dos filmes impressionados com bandas imunoquimioluminométricos e analisar a intensidade da banda por imagem dedicada software de análise (por exemplo,., ImageJ). </li></ol>

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The authors have nothing to disclose.

Porque mutações germinativas do gene BRCA2 chumbo ao aumento do risco de diversos tipos de câncer, incluindo o câncer feminino e masculino de mama, câncer de ovário, câncer de próstata, câncer de pâncreas e melanoma 3,4, uma série de estudos têm sido realizados para compreender a função biológica da proteína BRCA2. A maioria destes estudos são baseados em genética, principalmente devido a dificuldades técnicas na análise de uma proteína gigante como BRCA2. O método descrito aqui...

O gene BRCA2 (cancro da mama susceptibilidade gene-2) codifica para uma proteína supressora de tumor que desempenha um papel crucial na reparação de DNA quebras de cadeia dupla através da regulação da função da enzima recombinase Rad51 1. BRCA2 também tem sido implicado na modulação da transcrição e o controlo do ciclo celular em dois. Mutações da linha germinativa no gene BRCA2 induzir uma autossómica dominante susceptibilidade para cancro da mama e do cancro do ovário em mulheres e cancro da próstata em homens, assim como a predisposição para outros tipos de cancro 3,4. No entanto, apesar do aumento do risco de desenvolvimento de cancro, BRCA2 que suprimem ou reduzem a função BRCA2 pode tornar as células de cancro mais vulnerável a agentes quimioterapêuticos que provocam danos no DNA 5-7. Cânceres esporádicos apresentam uma baixa taxa de mutações BRCA2 (&lt;3%) que os níveis reduzidos de proteína BRCA2 foram detectados em alguns tipos de câncer, o que sugere que a proteína BRCA2podem ser perdidos durante a tumorigênese em cânceres esporádicos através de mecanismos não-mutacional-dependentes 7,8. Assim, é importante compreender as funções BRCA2 no contexto da biologia do cancro, bem como em outras configurações biológicos. Uma poderosa ferramenta utilizada para identificar novos funções de um gene em células de mamífero é silenciar a sua expressão. Como um exemplo, o silenciamento BRCA2 expressão numa variedade de células epiteliais normais levou recentemente à identificação de uma nova função de BRCA2 como regulador de resistência anoikis, um passo importante durante a aquisição de célula de cancro invasivo e metastático capacidade 9. Gene silenciamento pode ser alcançado através da introdução nas células ou de interferência (Si) ou ARN pequeno gancho de cabelo curto (sh) moléculas de RNA alvo do gene específico. A alta potência de siRNA e sua facilidade de uso tornam a ferramenta preferencial para silenciar experiências destinadas a ganhar novos insights sobre processos biológicos críticos umnd para identificar novos alvos terapêuticos. No entanto, knockdown eficiente da expressão do gene pode não ser facilmente possível para todos os genes e pode ser altamente variável dependendo do tipo de célula. Porque a diminuição dos níveis de proteína intracelular é o fenótipo mais relevante sob investigação, que é fundamental para quantificar o silenciamento do gene por análise de imunotransf erência do produto do gene. Com este respeito, a proteína BRCA2 apresenta um desafio adicional: sendo uma proteína grande (cerca de 390 kDa), dificuldades técnicas existem para análise bioquímica convencional, incluindo a imunotransferência. Nós relatamos aqui um protocolo otimizado para silenciamento eficiente do BRCA2 em linhas de células epiteliais humanas e para a detecção rápida e bem sucedida de proteína BRCA2 knockdown por immunoblotting análise.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BRCA2 siRNA</td>
			<td>Dharmacon</td>
			<td>L-003462-00</td>
			<td>SMARTpool: ON-TARGETplus BRCA2 siRNA</td>
		</tr>
		<tr>
			<td>Ctrl siRNA</td>
			<td>Dharmacon</td>
			<td>&#160;D-001810-10-05</td>
			<td>ON-TARGETplus Non-targeting Pool</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Reagent&#160;</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/13778075">13778075</a></td>
			<td>Transfection Reagent</td>
		</tr>
		<tr>
			<td>OPTI-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Medium for transfection procedure</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast</td>
			<td>Life Technologies</td>
			<td>EA0378</td>
			<td>Gel for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample buffer&#160;</td>
			<td>Life Technologies</td>
			<td>NP0007</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Sample Reducing agent (10x)</td>
			<td>Life Technologies</td>
			<td>NP0004</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Antioxidant</td>
			<td>Life Technologies</td>
			<td>NP0005</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>HiMark Pre-stained protein standard</td>
			<td>Life Technologies</td>
			<td>LC5699</td>
			<td>Prestained marker for gel electrophoresis</td>
		</tr>
		<tr>
			<td>XCell SureLoc Mini-Cell&#160; System</td>
			<td>Life Technologies</td>
			<td>EI0002</td>
			<td>Equipment for protein electrophoresis/transfer</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate SDS Running Buffer</td>
			<td>Life Technologies</td>
			<td>LA0041</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Life Technologies</td>
			<td>NP0006-1</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>BRCA2 H-300 rabbit polyclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8326</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Beta-tubulin monoclonal antibody</td>
			<td>Sigma Aldrich</td>
			<td>T4026-100UL</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Skim Milk powder</td>
			<td>Sigma Aldrich</td>
			<td>70166</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico/Femto Chemiluminescent Substrate</td>
			<td>Pierce Thermo Scientific</td>
			<td>34080/34096</td>
			<td>Chemiluminescence system</td>
		</tr>
		<tr>
			<td>PNT1A cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>95012614</td>
			<td>PNT1A human, normal prostate epithelium immortalized with SV40</td>
		</tr>
		<tr>
			<td>Nthy cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>90011609</td>
			<td>Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells</td>
		</tr>
		<tr>
			<td>Cell Proliferation Kit I (MTT)</td>
			<td>Roche</td>
			<td>11465007001</td>
			<td>Kit for cell proliferation assays</td>
		</tr>
	</tbody>
</table>

silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells

O silenciamento do BRCA2 proteína supressora de tumor e sua detecção por análises bioquímicas convencionais representam um grande desafio técnico, devido ao grande tamanho da proteína BRCA2 humano (cerca de 390 kDa). Relatamos modificações de transfecção e immunoblotting protocolos padrão de siRNA para silenciar BRCA2 e detectar a proteína humana endógena BRCA2, respectivamente, em linhas de células epiteliais humanas. Passos-chave incluem um elevado siRNA à relação de reagente de transfecção e duas rodadas subseqüentes de transfecção siRNA dentro do mesmo experimento. Usando estas e outras modificações introduzidas no protocolo padrão que consistentemente conseguir mais do que 70% silenciamento do gene BRCA2 humano, tal como avaliado por análise de imunotransf erência com anticorpos anti-BRCA2. Além disso, a desnaturação dos lisados ​​celulares a 55 ° C em vez da convencional de 70-100 ° C e outras optimizações técnicas do procedimento de imunotransferência permitir a detecção de proteína intacta, mesmo BRCA2 when quantidades muito pequenas de material de partida estão disponíveis ou quando os níveis de expressão de proteína BRCA2 são muito baixos. Silenciamento eficaz de BRCA2 em células humanas oferece uma estratégia terapêutica valiosa para interromper a função BRCA2 em células intactas com BRCA2, incluindo as células tumorais, para examinar novas vias moleculares e as funções celulares que podem ser afectadas por mutações em tumores BRCA2 patogénicos. Adaptação deste protocolo para silenciamento e análise de outras proteínas &quot;grandes&quot; como BRCA2 eficiente deve ser facilmente alcançável.

Antes de prosseguir com um ensaio biológico / bioquímicas para examinar o efeito de silenciamento BRCA2 em funções celulares, o primeiro passo é para confirmar a especificidade do silenciamento usando um siRNA mexidos (não-alvo siARN) lado a lado com BRCA2 siRNA (Figura 1A ). É importante a realização de um segundo turno de transfecção com o siRNA um elevado rácio de siRNA / transfecção para obter uma elevada eficiência de silenciamento BRCA2. Com efeito, a realização ...

Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Silenciamento de<em&gt; BRCA2</em&gt; Identificar funções biológicas regulada-BRCA2 romance em células humanas de cultura

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

silencing-brca2-to-identify-novel-brca2-regulated-biological

Research

JoVE Journal

Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

9.0K visualizações.  National Research Council. Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. 

Vídeo: Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Imagens ao vivo de vesículas com núcleo denso na Primária Cultivadas neurônios do hipocampo

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Biologia

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Uma análise quantitativa para o Estudo de Proteína: DNA interações, Discover Transcricionais Reguladores de Expressão Gênica e identificar novos agentes anti-tumorais

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualização do retículo endoplasmático Subdomains em células cultivadas

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Captura de Espectrometria de Massa Compound - uma ferramenta poderosa para identificar novos c-di-GMP efetoras Proteínas

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Screening siRNA identificar Ubiquitin e ubiquitina-como reguladores do sistema de caminhos biológicos em células de mamíferos cultivadas

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina microinjeção no peixe-zebra ao modelo de lesão renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Uma interface de usuário gráfica para rastreamento assistido por software da concentração de proteínas em protrusões celulares dinâmicas

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Medição da captação de glicose e resposta à estimulação de insulina em miotubos primários humanos cultivados in vitro

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...