Agradecemos ao Instituto Nacional de Saúde (1R21CA138373 e Stand Up to Cancer (SU2C) para suporte financeiro deste trabalho. HY é grato para o Prêmio Elion 2009 da Associação Americana de Pesquisa do Câncer, o Prêmio Acadêmico Kimmel do Sidney Kimmel Fundação para a Cancer Research (SKF-08-101), e da National Science Foundation Faculdade Prémio Carreira precoce (NSF0954819).

1. Considerações sobre a clonagem <ol><li> Oligonucleotides comercialmente preparado representando o TMD de interesse ladeado por NheI e sítios de restrição BamHI e 5&#39;-fosforilada pode ser ligada em pTox7 (modificado em nosso laboratório pela inserção de um par de base logo após a restrição BamHI 14 sites) (Figura 3) digerida seqüencialmente com BamHI e NheI. Um oligonucleotídeo exemplo é mostrado abaixo: 5&#39;ctagcTMDSEQUENCEg3 &#39; 3 &#39;gTMDSEQUENCEcctag5&#39; </li></ol> A seqüência deve ser DTM 24/12 resíduos (seqüências mais curtas será presumivelmente alongada por resíduos hidrofóbicos vetor codificado). , A fim de investigar a interface, quatro variantes do projeto DTM devem ser criados onde inserções resíduo seqüencial e concomitante resíduo resultado eliminações em rotação da DTM em relação ao ToxR 15,16. Finalmente, a concentração deve ser arabinose variou entre 0,001 e 0,01% (w / v) para identificar a concentração onde as diferenças máximo em β-galactosidase sinais entre diferentes seqüências de DTM são observados; testar diferentes níveis de expressão é recomendado para identificar as condições sob as quais afinidades diferentes podem ser distinguidos melhor. Além de arabinose e antibióticos, 0,4 mM IPTG pode ser usado para melhorar as diferenças de afinidades entre DTM diferentes. A medição ToxR deve ser realizada pelo menos em quatro vias. Todo o procedimento deve ser repetido pelo menos três vezes com diferentes transformações plasmídeo. 2. Crescimento de culturas bacterianas <ol><li> Suavemente descongelar FHK12 células competentes (200 mL) no gelo e transferir para um tubo de cultura de 15 ml. Adicionar DNA plasmídeo (200 ng) e incubar as células no gelo por 30 min. </li><li> Choque térmico células de incubação por 90 s em 42 ° C, seguido de incubação em gelo por 2 min. </li><li> Adicionar SOC media (800 mL) e incubar as amostras a 37 ° C com agitação (300 rpm) para um h. </li><li> Inocular 5 ml de mídia LB com cloranfenicol (30 mcg / ml) e arabinose (0,0025% w / v), com 50 ul da mistura transformação em tubos de 15 ml de cultura em triplicado. Incubar as amostras a 37 ° C com agitação (300 rpm) por 20 h. (Alternativamente 5 mL da cultura pode ser usada para inocular 100 ml de meio em uma placa de 96 poços. Este método é útil quando se lida com um grande número de amostras, apesar de erros vai ser um pouco maior. Para evitar a evaporação, o que levaria ao erro, preencha os poços ultraperiféricas com a mídia, mas não usá-los para as amostras. Finalmente, dê um duplo-wrap a articulação entre a tampa ea placa com parafilme). </li></ol> 3. Medição de β-galactosidase Atividade <ol><li> Pré-aqueça o leitor de placas a 28 ° C. </li><li> Transferir o Z-buffer em um reservatório com uma ponteira de grande porte, certificando-se de levar apenas a camada (aquoso) superior. Transferência de 100 l de Z-buffer/chloroform preparada para os poços de uma placa de 96 poços. Transferir 5 mL de cada cultura para os poços da placa em quatro vias. Omitir a cultura a partir de quatro poços, que servirá como o branco. </li><li> Medir o OD 595 da placa para determinar a densidade de células. </li><li> Adicionar 50 ul de Z-buffer/SDS a todos os poços da placa. Agite a placa na placa-leitor por 10 min para Lyze as células. Certifique-se que as suspensões de células são claras depois da lise e repita o passo tremendo, se necessário. Lise incompleta sugere a Z-buffer/chloroform não estava preparada. </li><li> Adicionar 50 ul preparada Z-buffer/ONPG a todos os poços e voltar a placa para o leitor de placas e medir OD 405 a cada 30 s por 20 min. </li><li> Calcular β-galactosidase atividade usando a seguinte equação (lembrando-se de subtrair o branco). A proporção de OD 405 / min deve ser calculado usando todos os pontos de dados na faixa de 405 OD 0,0-1,0 usando um ajuste do modelo linear. <img src="files/ftp_upload/2721/2721eq1.jpg" alt="Equação 1" /> Miller unidades diferentes, por vezes, quando gravadas em dias diferentes. Portanto, uma construção de referência como o GPA deveria ser medido em cada ensaio. Seus valores podem ser utilizados para a normalização dos valores ToxR. </li></ol> 4. Controle para a expressão da proteína <ol><li> Executar Western blotting para verificar mesmo a expressão da proteína entre os construtos. Combine 50 ul das culturas triplicado e centrifugar (2000 rpm, 4 min) em microcentrífuga. Retire o sobrenadante por pipetagem e ressuspender o sedimento residual em 2 x de tampão de carregamento de amostra. </li><li> Load 7,5 mL em um gel padrão de 8% e realização de eletroforese a 125 V durante 1 min horas 5. Após a transferência, incube com anticorpos anti-MBP HRP-conjugado e visualizar, a proteína quimérica é observado em aproximadamente 70 kDa com alguns produtos de degradação, por vezes visto em torno de 48 kDa. MBP endógena também é observado em 45 kDa (Figura 5). </li></ol>5. Controle de Inserção Membrana adequada A linhagem de células deficientes em proteína de ligação a maltose é utilizado para avaliar a inserção adequada da membrana da construção TMD quimérico. Quando cultivada em meio mínimo com maltose como a única fonte de carbono, apenas células que expressam uma membrana-integrante do produto expressão com maltose binding protein corretamente localizado à periplasma são capazes de crescer. <ol><li> PD28 transformar células (como descrito para FHK12 células) e inocular 2 ml de meio LB. Cultivar as células a 37 ° C com agitação (300 rpm) durante a noite. </li><li> Agregar as células por centrifugação a 3500 rpm, 10 min, 4 ° C e lavar por ressuspensão em PBS (2 ml) por pipetagem gentil com uma ponta grande ou vórtex gentil. Agregar as células (como acima), lavar com PBS por uma segunda vez, pelota e, finalmente, ressuspender em PBS (1 ml). </li><li> Use 25 mL de células em suspensão para inocular 5 ml meio mínimo em triplicado e incubar a 37 ° C com agitação (300 rpm). Tome OD 595 leituras entre 15-25 h, aproximadamente a cada duas horas, transferindo 200 mL de cada amostra em uma placa de 96 poços e leitura usando a placa-reader. </li></ol> 6. Resultados representativos: Um exemplo do uso do ensaio repórter ToxR transcricional para analisar a propensão oligomerização de domínios transmembrana mostrado na Figura 4. Anteriormente nós investigamos a oligomerização de domínios transmembrana da membrana multispanning-integrante da membrana latente proteína-proteína 1 (LMP-1) por várias técnicas, incluindo ToxR 14 domínios transmembrana cinco (TM5) foi mostrado para exibir uma forte propensão a oligomerize.; Isso é demonstrado pelo alto Unidades Miller, comparável ao controle positivo, o GPA, uma seqüência bem estabelecida dimerizing. Uma mutação deletéria no TM5, D150A, reduz a capacidade da seqüência a oligomerize. LMP-1 TM1 não significativamente oligomerize e exibe um sinal de unidade muito baixo Miller, um pouco acima do sinal para branco, não transformados FHK12 células. <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="Figura 1" /> Figura 1 dos desenhos animados. Retratando o ensaio repórter ToxR. Domínio transmembrana (DTM) impulsionado resultados oligomerização na dimerização de ToxR e ativação da transcrição LacZ. O produto do gene de LacZ, β-galactosidase pode ser quantificada como uma medida da propensão de um TMD de oligomerize. <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="Figura 2" /> Figura 2. A clivagem hidrolítica de ONPG pela β-galactosidase resulta na produção da espécie luz absorvendo o nitrophenolate (ONP). <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="Figura 3" /> Figura 3. Mapa Plasmid de pToxR7. <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="Figura 4" /> Figura 4. Representante ToxR ensaio repórter transcricional analisar a propensão oligomerização de membrana latente proteína-1 domínios transmembrana. Domínio transmembrana 5 (TM5) oligomerizes fortemente, enquanto um domínio transmembrana (TM1) exibe apenas uma interação fraca. D150A mutação no TM5 reduz significativamente sua capacidade de oligomerize. GPA é incluída como uma seqüência de controle positivo para a dimerização forte. Em branco representa untransformed FHK12 células. <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="Figura 5" /> Figura 5. Western blot para expressão da proteína. <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="Figura 6" /> Figura 6. PD28 ensaio de complementação para controlar a inserção da membrana correto para o periplasma. Controle negativo representa uma construção deficiente em proteína de ligação maltose.

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O ensaio ToxR repórter transcricional é uma maneira fácil de identificar seqüências transmembrana com potencial para oligomerize. Uma vez que as interações estão ocorrendo dentro da membrana bacteriana interior, este ensaio contorna os problemas associados com a validade de sistemas de estudar na membrana-miméticos ambientes. Dado que a clonagem de DTM múltiplas em um único plasmídeo pode ser facilmente feito em paralelo eo ensaio completo pode ser realizado em formato de 96 poços da placa, este ensaio pode...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Enzima de restrição BamHI </td><td> Invitrogen </td><td> 15201023 </td><td> Enzimas Invitrogen foram encontrados para ser mais eficiente do que fornecedores alternativos </td></tr><tr><td> NheI enzima de restrição </td><td> Invitrogen </td><td> 15444011 </td><td> Enzimas Invitrogen foram encontrados para ser mais eficiente do que fornecedores alternativos </td></tr><tr><td> 15 mL tubos de cultura </td><td> Fisher Scientific </td><td> 14-956-1J </td><td></td></tr><tr><td> SOC media </td><td> Teknova </td><td> S0225 </td><td> Feitas até o volume adequado e esterilizado em autoclave. </td></tr><tr><td> Media LB </td><td> Sigma-Aldrich </td><td> L7275 </td><td> Feitas até o volume adequado e esterilizado em autoclave. </td></tr><tr><td> Cloranfenicol </td><td> Sigma-Aldrich </td><td> CO 3 78 </td><td> Solução estoque de 30 mg / mL em etanol armazenados em freezer </td></tr><tr><td> Arabinose </td><td> Fluka </td><td> 10839 </td><td> Solução estoque de 2,5% (w / v) em água armazenado em freezer </td></tr><tr><td> Na 2 HPO 4 </td><td> Sigma-Aldrich </td><td> S9390 </td><td></td></tr><tr><td> NaH 2 PO 4 </td><td> Sigma-Aldrich </td><td> S9638 </td><td></td></tr><tr><td> KCl </td><td> Mallinckrodt Chemicals </td><td> 6858-06 </td><td></td></tr><tr><td> MgSO 4 .7 H 2 O </td><td> Sigma-Aldrich </td><td> 63138 </td><td></td></tr><tr><td> Dodecilsulfato de sódio (SDS) </td><td> Sigma-Aldrich </td><td> L6026 </td><td></td></tr><tr><td> 2-nitrofenil β-D-galactopiranosídeo (ONPG) </td><td> Sigma-Aldrich </td><td> 73660 </td><td></td></tr><tr><td> Z-buffer </td><td></td><td></td><td> 16,1 g Na 2 HPO 4 5.5g NaH 2 PO 4 0,75 g KCl 0.246g MgSO 4 Até perfazer 1 l, pH 7,0 </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200 mL β-mercaptoetanol, 2 mL de clorofórmio, faz até 20 mL com Z-buffer. Vortex por 1 min centrífuga, por 1 min a 800 rpm. Faça frescos para cada prato. </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160 mg SDS dissolvido em 10 mL Z-buffer </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 40 mg ONPG em 10 mL Z-buffer. Faça frescos para cada placa </td></tr><tr><td> β-mercaptoetanol </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Anti-MBP anticorpo monoclonal (HRP conjugado) </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td> Meio mínimo com maltose </td><td></td><td></td><td> 1 x sais M9, maltose 0,4%, 1 mg / mL de tiamina, 2 mM MgSO 4 </td></tr><tr><td> De 96 poços da placa de fundo chato </td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td> Leitor de placas- </td><td> Beckman Coulter </td><td> DTX880 Detector Multimodo </td><td></td></tr><tr><td> Banho de água </td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td> Agitação incubadora </td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

A visão simplista de domínios transmembrana da proteína como meramente âncoras em bicamadas de fosfolipídios foi há muito refutada. Em muitos casos, membrana que mede as proteínas têm evoluído mecanismos altamente sofisticados de ação. Uma maneira 03/01 em que as proteínas da membrana são capazes de modular as suas estruturas e funções é por contato direto e específico de hélices hidrofóbicas, formando oligômeros transmembrana estruturado 4,5. Recentes Grande trabalho centrou-se na distribuição de aminoácidos preferencialmente encontrados no ambiente de membrana, em comparação com solução aquosa e as diferentes forças intermoleculares que a associação de unidade de proteína. 6,7 No entanto, os estudos de reconhecimento molecular no domínio transmembrana das proteínas ainda fica atrás dos solúvel em água regiões. Um grande obstáculo continua: apesar da especificidade e afinidade notável que oligomerização transmembrana pode atingir de 8 a medição direta de sua associação é um desafio. Metodologias tradicionais aplicadas ao estudo da função integrante da membrana da proteína pode ser dificultada pela insolubilidade inerente das sequências em análise. Percepções biofísicos adquirida com a estudar peptídeos sintéticos representando domínios transmembrana pode fornecer informações estruturais úteis. No entanto, a relevância biológica da micelar detergente ou sistemas de lipossomas utilizados nestes estudos para imitar membranas celulares é muitas vezes questionada; fazer peptídeos adotar uma estrutura semelhante à nativa nessas condições e não o seu comportamento funcional refletem verdadeiramente o modo de ação dentro de uma membrana nativa ? A fim de estudar as interações de seqüências transmembrana em bicamadas de fosfolipídeos naturais, o laboratório de ensaios desenvolvidos Langosch repórter ToxR transcricional. 9 domínios transmembrana O de interesse é expressa como uma proteína quimérica, com ligação às proteínas maltose de localização para o periplasma e ToxR a apresentar um relatório do nível de oligomerização (Figura 1). Na última década, vários outros grupos (por exemplo, Engelman, DeGrado, Shai) otimizados e aplicados neste ensaio repórter ToxR. 10-13 Os ensaios ToxR vários tornaram-se um padrão-ouro para testar interações proteína-proteína em membranas celulares. Nós aqui demonstrar uma operação típica experimental realizado em nosso laboratório que segue protocolos desenvolvidos principalmente por Langosch. Este método de aplicação geral é útil para a análise de transmembrana associação auto-domínio em E. coli, onde β-galactosidase de produção é utilizado para avaliar a propensão oligomerização DTM. Após a DTM induzida por dimerização, ToxR se liga ao promotor ctx causando aumento da regulação do gene LacZ para β-galactosidase. A leitura colorimétrica é obtida pela adição de ONPG às células lisados. Clivagem hidrolítica de ONPG pela β-galactosidase resulta na produção da espécie luz absorvendo o nitrophenolate (ORA) (Figura 2).

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Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Um procedimento eficiente para avaliar a propensão oligomerização de domínios de uma única passagem transmembrana (DTM) é descrito. Proteínas quiméricas que consiste na DTM fundido a ToxR são expressos em uma cepa de E. coli repórter. DTM induzida por oligomerização causas dimerização de ToxR, a ativação de transcrição e de produção da proteína repórter, galactosidase.

Transmembrana Propensão Oligomerização Domínio determinado pelo Assay ToxR

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

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15.2K visualizações.  University of Colorado at Boulder. Um procedimento eficiente para avaliar a propensão oligomerização de domínios de uma única passagem transmembrana (DTM) é descrito. Proteínas quiméricas que consiste na DTM fundido a ToxR são expressos em uma cepa de E. coli repórter. DTM induzida por oligomerização causas dimerização de ToxR, a ativação de transcrição e de produção da proteína repórter, galactosidase.

Vídeo: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Expressão e purificação do Fibrose Cística proteína transmembranar regulador da condutância na Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Biologia

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Purificação da fibrose cística condutância transmembrana Regulador proteína expressa em<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

Caracterização dos receptores acoplados à proteína por uma mobilização de cálcio Ensaio baseado em fluorescência

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Genome-wide proteína-proteína Interaction Triagem por proteína-fragmento de Complementação Assay (PCA) em células vivas

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Reconstituição de uma proteína transmembrana, a voltagem-Ion Channel, KvAP, em Gigante Unilamelares Vesicles para microscopia e de Patch Estudos Grampo

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Mapeamento óptica de Intra-Sarcoplasmic Reticulum Ca<sup&gt; 2+</sup&gt; E Potencial Transmembrane no Coração Langendorff-perfundidos

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Abordagens genéticas e bioquímicas para<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; Avaliação de oligomerização da proteína: A Case Study rianodina Receptor

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Estudo do retículo endoplasmático e mitocôndrias Interações por<em&gt; In Situ</em&gt; Proximidade Ligadura Ensaio em células fixas

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...