Este trabalho foi apoiado pela concessão NSERC 386634. Gostaríamos de agradecer aos membros do Laboratório Screaton para discussões úteis.

1. Preparação dos reagentes, placas, e Células <ol><li> Adicione 500 ml de tampão de lise: Tris 25 mM, pH 7,5, NaCl 150 mM, NaF 50 mM, 0,5 mM EDTA pH 8,0, 0,5% Triton X-100, 5 mM beta-glicerofosfato, 5% de glicerol. Armazenar a 4 ° C. Imediatamente antes da utilização, adicionar 1 mM de ditiotreitol (DTT), 1 mM de fluoreto de fenilmetil sulfonil (PMSF), e 1 mM de vanadato de sódio. Após este passo, PMSF não é necessária em qualquer tampão de lavagem. </li><li> Adicione 20 ml de cinase 10X: Tris Buffer de 200 mM de pH 7,5, 50 mM beta-glicerofosfato (FW 216), vanadato de sódio a 2 mM. Da alíquota em tubos de 1 ml e armazenar a -20 ° C. Imediatamente antes de usar, adicionar DTT 5 mM. </li><li> Adicione 20 mL de tampão H-ATP 10x: 300 uM de adenosina trifosfato (ATP), 66 mM de MgCl2, 33 mM de MnCl 2. alíquota para tubos de 1 ml e armazenar a -20 ° C. </li><li> Adicione 100 ml de 2x dodecil sulfato de sódio (SDS) de tampão de lise: 1,5 g de base Tris, 20 ml de glicerol, 30 ml de H 2 O. Dissolver e ajustar o pH para 6,8 com HCl. Adicionar 40ml de SDS a 10%, ajustar o volume para 100 ml. Adicionar 25 mg de azul de bromofenol. </li><li> Spot 4 ul de 25 ng / uL do plasmídeo de expressão de mamífero que codificam uma cinase de GST-14 por poço em placas de conjuntos de 96 poços e as placas de etiqueta em conformidade. Seal e congelar placas a -20 ° C até à sua utilização. </li><li> 1-2 dias antes da transfecção, as células HEK293T cultura em meio de Eagle modificado por Dulbecco completo (DMEM) + 10% de soro fetal de bovino (FBS) e antibióticos numa incubadora a 37 ° C com CO 2 suplementado (final de 5%). Passage com tripsina e não permitir que o estoque para se tornar&gt; 80% confluentes durante a expansão. Um mínimo de 6 x 10 7 células são necessárias para a tela (aproximadamente 3 x 15 cm pratos de células HEK293T em 80% de confluência). </li></ol> 2. Transfecção Nota: Consulte a Figura 1 para um fluxograma de protocolo inteiro. <ol><li> Se placas contendo plasmídeos quinase foram congelados, descongele em temperatura ambiente umaND, centrifugar a 1900 xg durante 3 minutos para recolher a humidade no fundo dos poços. </li><li> Misturar 8,6 ml de meio de soro reduzido (por exemplo, meio OPTI-MEM), com 312,7 ul de reagente de transfecção à base de lípidos. Deixe descansar por 5 min. </li><li> Adicionar 10 ul de meio de soro reduzido para cada cavidade utilizando um dispensador de líquido automático equipado com um pequeno volume de cassete. </li><li> Adiciona-se 10 uL por poço de reagente de forma a mistura de soro / transfecção reduzida a partir do passo 2.2 utilizando um dispensador de líquido automático equipado com um pequeno volume de cassete. Deixe descansar por 20 a 45 min. </li><li> Ressuspender as células 293T em 7,5 x 10 5 células / ml em 80 ml ​​de DMEM completo. Adicionar 100 ul de suspensão de células (isto é, 7,5 x 10 4 células) por poço usando um distribuidor de líquido automático equipado com uma cassete de volume padrão. </li><li> Verifique poços sob o microscópio para distribuição uniforme de células e retornar à incubadora durante 24 h. </li></ol> 3. pulldown de GST-cinase <ol><li> Faça fresh: 4 mM de solução pervanadato por mistura de 60 ul de 0,2 M de vanadato de sódio, com 540 mL de H 2 O. Numa segunda mistura tubo de 2,7 mL de peróxido de 30% e 1,4 ml de PBS. Adicionar as duas soluções em conjunto e deixe descansar por 15 min antes do uso. </li><li> Usando uma pipeta de canais múltiplos (não utilizar um distribuidor de líquido automatizado uma vez que cria demasiada turbulência no poço) dispensar 2 ul de 0,25 M de CaCl2 em cada poço, seguido de 2,5 mL do pervanadato preparado no passo 3.1. Incubar cada placa a 37 ° C durante 10 min e, em seguida, colocar em gelo. </li><li> Prepare 35 ml de tampão de lise adicionando DTT, PMSF, e vanadato de sódio, tal como indicado na Seção 1 (Preparação de Reagentes). </li><li> Mantendo as placas em gelo, remover o meio de cada poço utilizando um aspirador de vácuo. Imediatamente adicionar 50 ul / poço de tampão de lise arrefecido em gelo utilizando um dispensador de líquido automático equipado com uma cassete de padrão. Deixe descansar 30 min em gelo para lise (opcional: pode parar por aqui, se necessário, por meio de selagem platE e armazenar a -80 ° C. Para continuar, placas de descongelamento no gelo). </li><li> Chapas giram a 1900 xg durante 3 min a 4 ° C. </li><li> Raspe células de cada poço utilizando uma pipeta de canais múltiplos e para transferir todos os conteúdos apropriadamente marcada placas de 96 poços de fundo em V. Chapas giram a 1900 xg durante 10 min a 4 ° C. </li><li> Durante a rotação, encher placas revestidas com glutationa (um para cada placa de 96 poços) com 100 ul / poço de tampão de lise arrefecido em gelo (sem adição de PMSF) como uma lavagem. Mantenha as placas no gelo. </li><li> Remover placas de fundo em V da centrífuga. Um de cada vez, inverter as placas de glutationa sobre uma pia para esvaziar a solução de lise e blot em uma toalha de papel. Transferir tampão de lise a partir das placas de fundo em V para as placas de glutationa por inclinação da placa e utilizando uma pipeta de canais múltiplos, tendo o cuidado de não perturbar o sedimento no fundo. Cubra as placas e deixar em gelo durante mínimo 2 horas de se ligar. </li><li> Perto do fim do passo de ligação 2 h, preparar uma estação de trabalho radioactividade, assegurando tele precauções de segurança necessárias estão no lugar para o trabalho radioativo. Definir forno de hibridação a 30 ° C. </li><li> Preparar 200 ml de tampão de lise por adição de DTT e vanadato de sódio tal como indicado no ponto 1. PMSF não é necessária nesta fase. </li><li> Inverter as placas de glutationa sobre uma pia para sacudir a tampão de lise e blot em uma toalha de papel. Lavar os poços 3x com 100 ul de tampão de lise (sem adição de PMSF). Não deixe que os poços sentar seco - mantê-los na lavagem até que esteja pronto para prosseguir. </li><li> Preparar 55 ml de tampão de quinase 1x (KB) diluindo o estoque de 10x e adição de DTT, tal como indicado no ponto 1. Lavar as placas uma vez com 50 ul de 1x KB utilizando um dispensador de líquido automático equipado com uma cassete de volume padrão. Deixar 1x KB em poços até uma solução pronta é: <ol><li> Prepare a Solução A pela adição de 500 a 530 ug de o substrato de interesse, 500 ug de proteína básica de mielina (MBP), 2,65 ml de 10x KB, 13,25 ul de DTT 1 M, e H2O até 15,9 ml. </li></ol></li&gt; <li> Um de cada vez, inverter as placas sobre uma pia para remover 1x KB enxaguamento, seque sobre toalha de papel, e imediatamente adicionar 30 ul de uma solução utilizando um dispensador de líquido automático equipado com um pequeno volume de cassete. Mantenha as placas no gelo. </li><li> Preparar a solução B na área de trabalho a radioactividade por adição de 2,5 ml de 10x H-ATP, 500 uCi de 32 P ATP gama-, e H 2 O a 10 ml. <ol><li> Adicionar 20 ul de solução B por poço utilizando uma pipeta de repetidor que auxilia na mistura, devido à força de ejecção. Cobrir e incubar em 30 ° C forno de hibridação durante 30 min. </li></ol></li><li> Após 30 minutos, a transferência de volta para as placas de gelo. Adicionar 50 ul de tampão de lise 2x SDS para cada poço utilizando uma pipeta de canais múltiplos. Pode prosseguir neste ponto para a próxima etapa, ou selar as placas com folha de alumínio e armazenar a -20 ° C até conveniente. </li></ol> 4. corrida, coloração e géis de secagem Nota: Todo o trabalho deve ser realizado em uma área DESIGNAted para a radioactividade. <ol><li> Ligue forno de hibridação e ajustado para 85 ° C. Placas de descongelamento em temperatura ambiente. Uma vez forno atingiu a temperatura, placas de transferência para forno e incubar por 10 min para desnaturar amostras. </li><li> Carga 26 poços géis pré-moldados com 15 ul de cada reacção utilizando uma pipeta de canais múltiplos para preencher vários poços de uma só vez. Cuidados devem ser tomados para que todas as dicas alinhar com as correspondentes bem antes de adicionar amostras. Execute gel em 150 V. Não deixe que o corante de rastreio (linha azul) funcionar fora do fundo do gel como esta contém a ATP não incorporado. </li><li> Desmontar os géis e cortou a ATP não incorporado (linha azul), utilizando um bisturi ou borda reta como ele vai expor demais os filmes. Coloque os gels em recipientes rotulados e cubra com Coomassie mancha para 15 min. </li><li> Remover a mancha de Coomassie, brevemente enxaguar os géis com água, e adicionar destain solução. Destain os géis até que as proteínas são claramente visíveis. Uma banda para MBP e uma banda para o substrato deveser visível para cada amostra. </li><li> Para secar o gel: <ol><li> Corte uma folha grande de papel de filtro e coloque-o no secador. </li><li> Molhe uma folha de celofane em água destilada até que esteja lisa e sem rugas, e colocá-lo no topo do papel. </li><li> Coloque os géis em cima da folha de celofane, fazendo uma nota da ordem de géis. Molhar uma segunda folha de celofane e colocar na parte superior dos géis. </li><li> Rolar para fora todas as bolhas (um rolo de vedação placa funciona bem para isso) para uma superfície uniforme agradável. Fechar a aba, ligar o vácuo, e secar o gel durante 3 horas a 80 ° C. </li></ol></li><li> Uma vez géis estão secos, expô-los a filme XAR com um ecrã de intensificar o sinal. Enrole a fita com saran wrap ou um saco plástico e feche com fita adesiva para impedir a entrada de geada. Armazenar a cassete a -80 ° C durante a noite. </li></ol> 5. Desenvolver XAR Films <ol><li> No dia seguinte, remove cassete do congelador e deixar descongelar à temperatura ambiente. Desenvolver o filme emuma câmara escura usando um processador de filme de acordo com as instruções do fabricante. Nota: Examinar os filmes para provas de pares quinase-substrato. Uma segunda exposição mais longa pode também ser útil para detectar eventos de fosforilação fracas. </li></ol>

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The authors have nothing to disclose.

Uma vez que as publicações originais que descrevem a abordagem 14,15, a biblioteca original de 180 GST-quinases foi ampliada para 420 membros, ou ~ 80% do kinome proteína humana. Com a biblioteca ampliada, o protocolo conforme descrito leva 4-5 dias e, em seguida, 1-4 dias para desenvolver filmes (como necessário), o que pode ser encurtado através da utilização de imagiologia com fósforo e o aumento do sinal digital. Há várias etapas-chave em que os cuidados devem ser tomados (veja ...

Proteína modificações pós-traducionais (PTMs) são essenciais para a comunicação intracelular. Talvez o melhor estudada de todas as PTMs fosforilação é catalisada por proteínas quinases, que regulam uma miríade de funções de proteínas, incluindo a sua actividade bioquímica, localização subcelular, conformação, e a estabilidade. A identificação de sítios de fosforilação de proteínas alvo pode ser realizada através de mapeamento triptico fosfopéptido ou por técnicas-padrão de proteómica agora utilizando amostras enriquecidas para péptidos fosforilados 1,2. Enquanto três quartos do proteoma expressas são esperados para ser fosforilada 3 e um identificado 200.000 locais de fosforilação 5, com estimativas de até 1 milhão de 6, muitos deles não têm atribuído biologia, via de sinalização, ou proteína quinase. Enquanto identificação de locais fosforilados é relativamente simples, uma comparativamente maior desafio éidentificar a cinase cognato (s) que tem como alvo estes locais, um processo que nos referimos como mapeamento de quinase: substrato pares. Várias abordagens para a identificação de quinase: substrato pares foram descritas, começando com uma quinase de interesse e procurando os seus substratos, ou a partir de um substrato de interesse e a tentativa de encontrar um modificador quinase 7-11 experimentalmente ou computacionalmente 12. Para identificar quinases para um substrato fosforilado conhecido, bioinformática pode ser usado para identificar proteínas que contêm uma sequência conservada curto de aminoácidos que flanqueiam o resíduo fosforilado (o local de consenso), bem como a identificação de quinases que formam um complexo com o substrato precipitável. No entanto, estas abordagens são demorados e muitas vezes não cumprem com sucesso. Nós desenvolvemos uma abordagem funcional sistemático para identificar rapidamente quinases que fosforilam pode um determinado substrato 13. O ensaio tela produz excelente específicodade, com a seleção muito clara para os potenciais quinases cognatas. Dada a centralidade da fosforilação de sinalização biológica, a tela é útil para a descoberta em praticamente todos os celulares vias de sinalização 14-16. A tela envolve a realização de um ensaio de quinase em larga escala com uma biblioteca de proteínas quinases humanos. As cinases foram marcados com glutationa bacteriano S-transferase (GST) de proteínas e são purificados a partir de extractos de células de mamíferos, o que significa que as enzimas recombinantes - ao contrário do que as preparadas a partir de bactérias - são gerados na presença das proteínas quinases a montante muitas vezes necessários para a enzimas recombinantes para ter actividade in vitro. Com efeito, enquanto que a actividade da cinase de serina, treonina e tirosina necessária para a activação a jusante de quinase estão presentes na levedura 10, o genoma da levedura codifica 122 cinases de proteína, indicando que o kinome de mamífero, com mais de 500 genes 17, tornou-se significativamente mais complexa, a fim de regulate os processos originais para os organismos de ordem mais elevada. Além disso, o efeito de diferentes estímulos relevantes para a biologia celular e doença humana (tal como pequenas moléculas, factores de crescimento, hormonas, etc.) podem ser usados ​​para modular a actividade de quinase 14,15 num contexto apropriado.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lysis buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.1</td>
		</tr>
		<tr>
			<td>10x kinase buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.2</td>
		</tr>
		<tr>
			<td>10x M-ATP</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.3</td>
		</tr>
		<tr>
			<td>Human kinase plasmids</td>
			<td>Orfeome, Invitrogen, Origene</td>
			<td></td>
			<td>GST-tagged in house</td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Fisher Scientific</td>
			<td>CS003595</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Fisher Scientific</td>
			<td>SH3002201</td>
			<td>supplement with 100U/ml penicillin, 100ug/ml streptomycin, 10% fetal calf serum.</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Sanyo</td>
			<td>MCO-17AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm cell culture dishes</td>
			<td>Fisher Scientific</td>
			<td>877224</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Invitrogen</td>
			<td>22600-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated liquid dispenser</td>
			<td>Thermo Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>24073295</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>14072670</td>
			<td></td>
		</tr>
		<tr>
			<td>4mM pervanadate</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 3.1</td>
		</tr>
		<tr>
			<td>0.25 M CaCl2</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (20-200 uL)</td>
			<td>Labnet</td>
			<td>p4812-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (1-10 uL)</td>
			<td>Thermo Scientific</td>
			<td>4661040</td>
			<td></td>
		</tr>
		<tr>
			<td>V-bottom 6-well plates</td>
			<td>Evergreen Scientific</td>
			<td>290-8116-01V</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione coated 96-well plates</td>
			<td>Fisher Scientific</td>
			<td>PI-15240</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Biostad</td>
			<td>350355</td>
			<td></td>
		</tr>
		<tr>
			<td>GST tagged substrate</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myelin Basic Protein (MBP)</td>
			<td>Sigma</td>
			<td>M1891</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater pipette (1 mL)</td>
			<td>Eppendorf</td>
			<td>22266209</td>
			<td></td>
		</tr>
		<tr>
			<td>32P gamma-ATP</td>
			<td>Perkin Elmer</td>
			<td>BLU502Z500UC</td>
			<td></td>
		</tr>
		<tr>
			<td>2X SDS lysis buffer (100 mL)</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.4</td>
		</tr>
		<tr>
			<td>26-well precast TGX gels</td>
			<td>BioRad</td>
			<td>567-1045</td>
			<td>gel percentage required is dependent on the molecular weight of the substrate of interest</td>
		</tr>
		<tr>
			<td>Coomassie stain</td>
			<td>Made in house</td>
			<td></td>
			<td>0.1% Coomassie R250, 10% acetic acid, 40% methanol</td>
		</tr>
		<tr>
			<td>Coomassie destain</td>
			<td>Made in house</td>
			<td></td>
			<td>10% acetic acid, 20% methanol</td>
		</tr>
		<tr>
			<td>Labeled gel containers</td>
			<td>Made in house</td>
			<td></td>
			<td>Used plastic lids from empty tip boxes, just big enough to contain one gel</td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td>Fisher Scientific</td>
			<td>57144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellophane sheets (2)</td>
			<td>BioRad</td>
			<td>165-0963</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel dryer</td>
			<td>Labconco</td>
			<td>4330150</td>
			<td></td>
		</tr>
		<tr>
			<td>Double emulsion autoradiography film</td>
			<td>VWR</td>
			<td>IB1651454</td>
			<td></td>
		</tr>
		<tr>
			<td>Film cassette</td>
			<td>Fisher Scientific</td>
			<td>FBAC-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Intensifying screen</td>
			<td>Fisher Scientific</td>
			<td>FBIS-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealing rubber roller</td>
			<td>Sigma</td>
			<td>R1275</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of kinase-substrate pairs using high throughput screening

Nós desenvolvemos uma plataforma de rastreio para identificar proteínas quinases específicas de substratos fosforilados humanos que podem ser utilizados para elucidar novas vias de transdução de sinal. A nossa abordagem apresenta a utilização de uma biblioteca de proteínas quinases humano marcado com GST purificada e um substrato da proteína recombinante de interesse. Usámos esta tecnologia para identificar MAP / microtúbulo-regulação afinidade quinase 2 (MARK2) como a quinase para um local regulada por glucose, em reguladas pela CREB transcricional coativador 2 (CRTC2), uma proteína necessária para a proliferação das células beta, assim como a família de tirosina-quinases Axl como reguladores de metástases de células por fosforilação da proteína adaptadora ELMO. Descrevemos essa tecnologia e discutir como ela pode ajudar a estabelecer um mapa abrangente de como as células respondem a estímulos ambientais.

Os resultados representativos de uma tela são mostrados na Figura 2. 180 quinases foram rastreadas utilizando um substrato de péptido marcado com GST correspondente aos aa 268-283 da CRTC2, bem como a proteína básica clássico ensaio de quinase substrato de mielina (MBP). Apenas duas cinases, MARK2 e o MARK3-quinase altamente relacionados com o peptídeo fosforilado CRTC2. MBP é incluído como um controlo interno em todos os ensaios, uma vez que contém muitos resíduos fosforiláv...

Watch this Scientific Journal Video about Identification of Kinase-substrate Pairs Using High Throughput Screening at JoVE.com

Identification of Kinase-substrate Pairs Using High Throughput Screening

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identificação de pares Kinase-substrato utilizando High Throughput Screening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

identification-kinase-substrate-pairs-using-high-throughput

Research

JoVE Journal

Biology

8.1K visualizações.  Children's Hospital of Eastern Ontario Research Institute. Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Vídeo: Identification of Kinase-substrate Pairs Using High Throughput Screening

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

Alto rendimento Triagem e Biossensoriais com Fluorescente C. elegans Cepas

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Biologia

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

High Throughput Screening da atividade endoglucanase de fungos em Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimento cristalização de proteínas de membrana Usando o método Bicelle lipídico

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Alta capacidade mapeamento físico de cromossomos usando Automated<em&gt; In situ</em&gt; Hibridação

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

High-throughput screening para pequenas moléculas Moduladores de canais de potássio Interiores retificador

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secundária Previsão Estrutura Usando SHAPE de alta capacidade

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

De alta capacidade de Triagem funcional usando um caseiro Dual-brilho ensaio de luciferase

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Automated Modular High Throughput exopolissacï¿½ido Plataforma de Triagem Juntamente com Análise Carboidratos Fingerprint Highly Sensitive

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Multi-enzima Triagem Usando um de alta capacidade Enzyme genética Sistema de Triagem

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...