Esta pesquisa foi apoiada por doações da Deutsche Forschungsgemeinschaft GraKo1121, Hu1011/2-1 e SFB940 a SH

Nota: Informações detalhadas sobre tampões indicados no protocolo está disponível na Tabela 1. 1. Fixação de interações proteína-proteína por formaldeído Cross-ligando em células vivas <ol><li> Para começar a preparar meios de cultura, soluções de reserva e de tampão P1 <ol><li> Prepare 500 ml meio: O meio deve proporcionar condições que suportam a interação entre a proteína isca membrana e proteínas presas. Além disso, um experimento deve ser realizado sob condições em que se espera nenhuma interação (por exemplo, sem cross-linking). Nota: Nós comparamos interações proteína-proteína em bactérias estressados ​​e não-estressados. Portanto, podemos preparar 500 ml de Luria Bertani (LB) (Tabela 1) que complementar com um agente de indução de stress (por exemplo, NaCl a 0,5 M) num frasco de 2 L de Erlenmeyer. Para o controle, usamos meio LB normal com 0,17 M de NaCl (pH 7,0). </li><li> Preparar tampão Tris (20 mM Tris-HCl, pH 8,0) e 0,1 M de ácido etilenodiaminotetracético (EDTA), pH 8,0 solução de estoque (Tabela 1). </li><li> Prepare tampão P1. Dissolve-se a sacarose a uma concentração final de 0,5 M em tampão Tris. Esterilizar tampão P1 por filtração e armazená-lo a 4 ° C. </li></ol></li><li> Crescer as células que expressam a proteína de membrana de isco com uma fusão C-terminal de Strep-tag de um vector. Induzem a expressão de proteínas de membrana de isco durante um tempo suficiente. Nota: Foram induzir a expressão de proteínas de membrana bacterianas isca em fase-log precoce (A 600 = 0,3) pela adição de 250 ul de 1 M isopropil-β-D-tiogalactopiranósido (IPTG) para 500 ml de meio (concentração final: 0,5 mM) . Para permitir uma expressão suficiente, que incuba-se as bactérias até à fase de fim de registo (A 600 = 1,2). </li><li> Prepare-se para cada uma cultura de duas provetas de centrifugação com um volume mínimo de 300 ml de cada um e colocá-las sobre gelo. </li><li> Realize formaldeído cross-linking. CAUÇÃO: O formaldeído é altamente tóxico Recomendamos trabalhando sob uma coifa de segurança.. <ol><li> Transfira o recipiente de cultura sob uma coifa de segurança. Dividir cada amostra em duas amostras, uma omissão de reticulação (- X) e uma incluindo o formaldeído de reticulação (+ X). Adicionar 4 ml de solução de formaldeído a 37% e 250 ml de cultura para atingir uma concentração final de 0,6%. </li></ol></li><li> Transfira os recipientes de cultura para trás e cultivar células por mais 20 min como antes. </li><li> Preencha seus culturas dentro dos tubos de centrífuga sob uma coifa de segurança. Recolher as células por centrifugação a 3000 xg durante 30 min. </li><li> Elimine o sobrenadante com cuidado pipetando-lo sob uma coifa de segurança. Colete tóxico sobrenadante contendo formaldeído e descartá-la adequadamente. </li><li> A fim de atingir o rendimento ideal durante a preparação de proteínas de membrana, preparar esferoplastos primeiro. Aqui, nós fornecemos um protocolo para a formação de esferoplastos de bactérias Gram-negativas: <ol><li> Prepartampão e P2 (Tabela 1) a partir de pó liofilizado. Suavemente dissolver 2 mg de lisozima em 0,1 M de EDTA, pH 8, num tubo de 1,5 ml. Sempre preparar tampão P2 na hora para o dia do experimento. </li><li> Prepare Tris-buffer com inibidor de protease (P3 buffer). A 10 ml de tampão Tris, adicionar 0,1 ml de 1 M de fluoreto de fenilmetilsulfonilo (PMSF) (Tabela 1) para uma concentração final de 10 mM. Use o tampão imediatamente, para garantir o funcionamento adequado da PMSF como inibidor da protease. </li><li> Os sedimentos de células Ressuspender em 10 ml de tampão Tris com inibidor de protease e solução de transferência para um tubo de 15 ml. </li><li> Adicionar 1 ml de tampão P2 e incubar em gelo durante 30 min. </li><li> Recolhe esferoplastos por centrifugação a 3000 xg durante 30 minutos e descartar o sobrenadante cuidadosamente. </li></ol></li><li> Incubar as pelotas de esferoplastos a noite a -20 ° C. </li></ol> 2. Purificação de Strep-tag Membrane Protein (Bait) <ol><li> Coloque esferoplasto pellet no gelo. &lt;/ Li&gt; <li> Durante o tempo dos peletes de esferoplastos descongelar em gelo, 10 ml de preparar P3 tampão para cada preparação de esferoplastos. Suavemente dissolver 1 mg ADNasel em 10 ml de tampão Tris. Adicionar 0,1 ml de 1 M de PMSF imediatamente antes da utilização. </li><li> Ressuspender o sedimento em 6 ml P3 preparada de fresco. Sonicar a amostra quatro vezes durante 1 min de forma contínua em gelo com 1 min de pausa entre cada explosão, de forma a interromper os esferoplastos. </li><li> Centrifugar a amostra a 10.000 xg durante 10 minutos para colher os detritos celulares. Transferir o sobrenadante com uma pipeta para um tubo de ultracentrifugação. </li><li> Sedimentar a fracção de membrana a 100.000 xg durante 30 min. Lavar o sedimento cuidadosamente em Tris-tampão sem dissolver. Seque o tubo com um lenço de papel (por exemplo, lenços de papel). Evitar perturbar o sedimento, em qualquer momento. </li><li> Ressuspender o sedimento (= membranas) cuidadosamente em 1 ml de tampão Tris. Use uma alíquota de 20 ul para determinar a concentração de proteína pelo ensaio de BCA por exemplo. Neste passo, as membranas podem ser congelados em choque liquid azoto e armazenada a -80 ° C. </li></ol> 3. Purificação de Strep-tag Membrana Isca proteína e SDS-PAGE <ol><li> Normalizar a concentração de proteína da fracção de membrana de 5 mg / ml com tampão Tris. Tome 2,5 ml da fracção de membrana (5 mg / ml) num tubo de ultracentrifugação e adicionar 0,25 ml de 20% Triton X-100 para se atingir uma concentração final de 2%, a fim de solubilizar as proteínas da membrana. Adicionar uma haste de micro-magnético e agita-se em gelo durante 1 hr. Nota: Apesar de, em geral, temos bons resultados com o Triton X-100 como detergente, pode ser útil para mudar o detergente para otimização. A este respeito, temos os melhores resultados com os detergentes utilizados para a purificação funcional da respectiva proteína isco membrana. </li><li> Embora a realização do passo 3.1, preparar 50 ml de tampão W e 5 ml de tampão E (Tabela 1). Encha-se 10 mL de 5x tampão W concentrado para 50 ml e adicionar 150 ul de 20% de Triton X-100. <ol><li> Encha-se 1 ml de 5x buffer E concentrado para 10 ml e adicionar 30 ul de 20% de Triton X-100. Equilibrar um Strep-Tactin coluna de fluxo de 1 ml superfluxo gravidade com 8 ml de tampão de W. Nota: É essencial para adicionar o detergente utilizado para a solubilização em uma concentração de 10 vezes acima da concentração micelar crítica (CMC) em qualquer solução durante o processo de purificação. </li></ol></li><li> Retirar a vareta micro-magnético a partir da amostra de solubilização e de ultracentrifugação a 100000 xg durante 30 minutos, para sedimentar a fracção de membrana insolúvel. Retirar o sobrenadante utilizando uma pipeta para purificação adicional. </li><li> Carrega-se o sobrenadante para a coluna. Executar a coluna só com fluxo por gravidade. Lavar a coluna com 5 ml de tampão W. Repetir esta etapa de lavagem 5x. </li><li> Eluir as proteínas de membrana de isca com 1 ml de tampão E. Repita este passo de eluição de 4x. </li><li> Concentra-se as fracções de eluição 2, 3 e 4, até 300 ml, com uma unidade de filtração centrífuga. </li><li> Misturar 200 ul de cada amostra com 50 mL de 5x de SDS-PAGCorante de carga de E. Divida cada preparação em 125 mL alíquotas. Ferver uma alíquota de cada preparação durante 20 min a 95 ° C, para reverter ligações cruzadas de formaldeído. Deixe as amostras arrefecer para a temperatura ambiente durante pelo menos 10 min, em cima da bancada. </li><li> Carga de 30 ul de cada amostra para uma única pista de um gel de poliacrilamida-gel adequado para imunoblotting. Use um marcador prestained peso molecular para uma melhor orientação e executar SDS-PAGE 10. </li></ol> 4. Análise Imunoblot para confirmar parceiros de interação <ol><li> Uma vez que o passo 3.8 for concluída, proteínas de transferência para uma membrana de nitrocelulose por exemplo semidry. </li><li> Bloquear a membrana para evitar a marcação não específica. Preparar o tampão de bloqueio por pesagem de albumina sérica bovina (BSA) para atingir o peso de 3% por volume em soro fisiológico tamponado com Tris suplementado com uma concentração final de 0,05% de Tween-20 (TBS-T). Utilizar um volume suficiente de tampão de bloqueio para cobrir a membrana. Bloquear a membrana durante 1 hora a RT. </li><li> Diluir umaª incubar o primeiro anticorpo específico da proteína presa como de costume. Usar um anticorpo ligado a HRP como o segundo anticorpo. </li><li> Desenvolver a imunotransferência utilizando um kit de detecção quimioluminescente com alta sensibilidade. Monitorar o sinal através de um procedimento de processamento de filmes clássicos ou equipamentos de imagem digital. </li></ol> 5. NanoLC-ESI-MS/MS de alta resolução Experimentos para identificar parceiros de interação <ol><li> No caso em que nenhum anticorpo específico está disponível para a proteína presa ou parceiros de interacção desconhecidos devem ser identificadas, utilizar a espectrometria de massa (MS) para a identificação. A fim de evitar a contaminação da queratina, usar géis precasted para a separação de proteínas. </li><li> Prata manchar a SDS-PAGE utilizando um kit de coloração MS-compatível de acordo com o protocolo do fabricante. Execute todas as etapas de coloração e lavagem de tanques de vidro. </li><li> Especiais de Consumo respectivas bandas e analisar estes de alta resolução por LC / MS 9. </li></ol>

<ol>
	<li>Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E. L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+transmembrane+protein+topology+with+a+hidden+markov+model:+application+to+complete+genomes.">Predicting transmembrane protein topology with a hidden markov model: application to complete genomes.</a> J. Mol. Biol. 305, 567-580 (2001).</li><li>Bakheet, T. M., Doig, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+and+identification+of+human+protein+drug+targets.">Properties and identification of human protein drug targets.</a> Bioinformatics. 25, 451-457 (2009).</li><li>Yildirim, M. A., Goh, K. I., Cusick, M. E., Barabasi, A. L., Vidal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-target+network.">Drug-target network.</a> Nat. Biotechnol. 25, (2007).</li><li>Daley, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assembly+of+membrane+proteins+into+complexes.">The assembly of membrane proteins into complexes.</a> Curr. Opin. Struct. Biol. 18, 420-424 (2008).</li><li>Jung, K., Fried, L., Behr, S., Heermann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histidine+kinases+and+response+regulators+in+networks.">Histidine kinases and response regulators in networks.</a> Curr. Opin. Microbiol. 15, 118-124 (2012).</li><li>M&#252;ller, V. S., Jungblut, P. R., Meyer, T. F., Hunke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-SPINE:+An+improved+method+to+identify+protein-protein+interaction+partners+of+membrane+proteins+in+vivo.">Membrane-SPINE: An improved method to identify protein-protein interaction partners of membrane proteins in vivo.</a> Proteomics. 11, 2124-2128 (2011).</li><li>Herzberg, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPINE:+A+method+for+the+rapid+detection+and+analysis+of+protein-protein+interactions+in+vivo.">SPINE: A method for the rapid detection and analysis of protein-protein interactions in vivo.</a> Proteomics. 7, 4032-4035 (2007).</li><li>Sutherland, B. W., Toews, J., Kast, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+formaldehyde+cross-linking+and+mass+spectrometry+in+the+study+of+protein-protein+interactions.">Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions.</a> J. Mass Spectrom. 43, 699-715 (2008).</li><li>Hernandez, P., M&#252;ller, M., Appel, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+protein+identification+by+tandem+mass+spectrometry:+Issues+and+strategies.">Automated protein identification by tandem mass spectrometry: Issues and strategies.</a> Mass Spectrom. Rev. 25, 235-254 (2006).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Hunke, S., Keller, R., M&#252;ller, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+by+the+Cpx-envelope+stress+system.">Signal integration by the Cpx-envelope stress system.</a> FEMS Microbiol. Lett. 326, 12-22 (2012).</li><li>Weber, R. F., Silverman, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cpx+proteins+of+Escherichia+coli+K12+:+Structure+of+the+CpxA+polypeptide+as+an+inner+membrane+component.">The Cpx proteins of Escherichia coli K12 : Structure of the CpxA polypeptide as an inner membrane component.</a> J. Mol. Biol. 203, 467-478 (1988).</li></ol>

Não temos nada a divulgar.

Análise Membrane coluna vertebral é uma abordagem bioquímica que permite que se confirmar e identificar este ponto parceiros de interação desconhecidos de proteínas de membrana. Membrana COLUNA combina in vivo cross-linking por formaldeído com purificação de uma proteína de membrana isca Strep-marcado. A combinação com imunotransferência facilita a confirmação de parceiros de interacção previstas (Figura 2). Além disso, a combinação com análise MS permite a identificação ...

Para entender a função de uma proteína que é essencial conhecer os seus parceiros de interação. Várias técnicas clássicas estão disponíveis para a identificação de parceiros de interacção de proteínas solúveis. No entanto, estas técnicas não são facilmente transferíveis para a membrana proteínas, devido à sua natureza hidrofóbica 4. Para ultrapassar esta limitação, foi desenvolvido o Strep-proteína de membrana interacção experimento (Membrane-SPINE) 6. Ele baseia-se no método da coluna vertebral, que era apenas adequado para proteínas solúveis 7. Benefícios membrana da coluna vertebral de duas vantagens do formaldeído agente de reticulação: primeiro, o formaldeído pode facilmente penetrar as membranas e, consequentemente, gera uma imagem precisa da interactoma de uma célula viva 8. Em segundo lugar, formaldeído ligações cruzadas pode ser revertida por ebulição 9. Aqui, estas duas vantagens são usados ​​para identificar não só IPP permanentes, mas também transientes de prote membranains 6. Em resumo, um Strep-tag está fundido com o terminal C da proteína de membrana integral isca. As células que expressam a proteína de membrana de isca são incubadas com formaldeído, que as ligações cruzadas atacam proteínas para a proteína isco membrana (Figura 1). Modificações de proteínas presas não são necessários. Em seguida, a fracção de membrana é preparado. Portanto, as proteínas da membrana são solubilizados por tratamento com detergente e isca proteínas são co-purificados com suas proteínas presas utilizando a purificação por afinidade. Subsequentemente, a ligação transversal é invertida por ebulição, e o isco e suas proteínas presa co-eluidas são separadas por SDS-PAGE. Finalmente, as proteínas de presa pode ser identificado por análise de imunotransferência ou espectrometria de massa.

<table border="0" cellpadding="0" cellspacing="0" width="878">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">37% Formaldehyde</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">4979.1</td>
			<td style="width:271px;">Should not be older than one year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DNaseI</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:137px;">DN25</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BCA protein assay kit</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">23225</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Micromagnetic rod</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">0955.2</td>
			<td style="width:271px;">5 mm in length, 2 mm in diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Triton X-100</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">6683.1</td>
			<td style="width:271px;">standard detergent for solubilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">n-Dodecyl-&#946;-maltoside</td>
			<td style="width:128px;">Glycon</td>
			<td style="width:137px;">D97002-C</td>
			<td style="width:271px;">the best detergent to solubilze CpxA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">1 ml Strep-Tactin superflow gravity flow column</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1207-050</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Strep-tag protein purification buffer set</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1002-001</td>
			<td style="width:271px;">Contains buffer W and buffer E</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Amicon Ultra-4 centrifugal filter</td>
			<td style="width:128px;">Millipore</td>
			<td style="width:137px;">UFC803024</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">SuperSignal West Pico Chemiluminescent substrate</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">34080</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">SilverQuest Silverstaining kit</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:137px;">LC6070</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">FireSilver Staining Kit</td>
			<td style="width:128px;">Proteome Factory</td>
			<td style="width:137px;">P-S-2001</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Ultracentrifuge</td>
			<td style="width:128px;"></td>
			<td style="width:137px;"></td>
			<td style="width:271px;"></td>
		</tr>
	</tbody>
</table>

membrane-spine: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo

As proteínas da membrana são essenciais para a viabilidade celular e, portanto, são alvos terapêuticos importantes 1-3. Desde que funcionam nos complexos de 4, métodos para identificar e caracterizar as suas interacções são necessárias 5. Para isso, desenvolvemos a membrana Strep-proteína experimento interação, chamada de membrana-espinha 6. Esta técnica combina in vivo de ligação cruzada utilizando o formaldeído reversível de reticulação com a purificação por afinidade de uma proteína de membrana de isca Strep-tag. Durante o procedimento, as proteínas presa reticulados são co-purificados com a proteína de membrana de isca e, subsequentemente, separado por ebulição. Assim, duas grandes tarefas podem ser executadas quando se analisa as interações proteína-proteína (IBP) de proteínas de membrana usando Membrane-espinha: primeiro, a confirmação de um parceiro de interação proposto por immunoblotting e, segundo, a identificação de novos parceiros de interação através da análise de espectrometria de massa . Além disso, mesmo low afinidade PPIs transientes são detectáveis ​​por esta técnica. Finalmente, Membrane-espinha é adaptável a praticamente qualquer tipo de célula, tornando-o aplicável como ferramenta de triagem para identificar poderoso PPIs de proteínas de membrana.

A análise da membrana COLUNA permite co-purificação de proteínas da membrana e transitoriamente interagindo parceiros proteicos. O co-purificação é conseguido usando o agente formaldeído cross-linking. Dois parâmetros são críticos para evitar inespecíficas ligações cruzadas: a concentração de formaldeído e o tempo de reticulação. A utilização suficientes, mas não excessivas de formaldeído pode ser facilmente controlada por imunotransferência. Complexos proteicos formaldeído reticulado pode ser s...

Watch this Scientific Journal Video about Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo at JoVE.com

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A abordagem bioquímica é descrita a identificação in vivo de interacções proteína-proteína (PPI), de proteínas de membrana. O método combina a proteína de ligação cruzada, de purificação por afinidade e espectrometria de massa, e é adaptável para quase qualquer tipo de célula ou organismo. Com esta abordagem, embora a identificação de IPP transientes torna-se possível.

Membrana-COLUNA: Uma Ferramenta bioquímicos na identificação de interações proteína-proteína de Proteínas de Membrana<em&gt; In Vivo</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

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Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

13.6K visualizações.  Universität Osnabrück.  A abordagem bioquímica é descrita a identificação in vivo de interacções proteína-proteína (PPI), de proteínas de membrana. O método combina a proteína de ligação cruzada, de purificação por afinidade e espectrometria de massa, e é adaptável para quase qualquer tipo de célula ou organismo. Com esta abordagem, embora a identificação de IPP transientes torna-se possível. 

Vídeo: Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Fabricação de Eletroquímica-DNA Biossensores para Detecção Reagentless de ácidos nucléicos, proteínas e pequenas moléculas

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Bioengenharia

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Visualizando Proteínas e Complexos Macromoleculares por Negative EM Stain: Preparação de Grid para Aquisição de Imagem

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinação da taxa de transporte de xenobióticos e nanomateriais através da placenta usando o<em&gt; Ex vivo</em&gt; Modelo de perfusão da placenta humana

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

Isolamento e caracterização de proteínas quimricas que expressam fc humanos usando adsorbers de membrana de proteína e um fluxo de trabalho simplificado

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Luminescência Ressonância transferência de energia para estudar mudanças conformacionais na membrana proteínas expressas em células de mamíferos

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluorescência-têmpera de um Fluoróforo encapsulado em lipossomas Near-infrared como ferramenta para<em&gt; In Vivo</em&gt; Imageamento óptico

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambientes sanduíche-como aproveitarar Telemóveis / interações materiais

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparação de 3D colágeno Géis e microcanais para o estudo das interações 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Nanotubos de DNA como uma ferramenta versátil para estudar semiflexível polímeros

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...