Este trabalho foi financiado pela R &amp; D Systems, Inc.

1. Preparação da Amostra <ol><li> Colete de lisados ​​não tratada, ligando tratada, inibidor ou ligante e células tratados seguindo as orientações na folha de dados matriz humana Phospho RTK. O tampão de lise foi otimizado para este kit. A substituição dos tampões de lise podem afectar o desempenho final da matriz. Para evitar a degradação proteolítica da amostra, adicionar 10 ug / ml de aprotinina, 10 ug / ml de leupeptina, pepstatina e 10 ug / ml de A para o volume de tampão de lise requerido para cada preparação de lisado de células fresca para cada utilização. </li><li> Lysate concentração deve ser medido utilizando um ácido bicinconínico (BCA) de ensaio. Concentração da amostra pode ser empiricamente ajustada para uma sensibilidade óptima e de fundo baixo. Uma gama de 100-300 ng de lisado é recomendado como um ponto de partida inicial. </li><li> Congelamento / descongelamento de amostras devem ser evitados e armazenamento adequado de lisados ​​é necessário para o desempenho ideal. Descongelar as amostras congeladas lisado em gelo. </li></ol><ol><li> Trazer todos os componentes do kit para a temperatura ambiente antes do uso. Para evitar contaminação, usar luvas durante a realização dos procedimentos. </li><li> Pipetar 2,0 ml de tampão de matriz 1 em cada poço do prato de 4 Bem-Multi a ser utilizado. Tampão de matriz 1 serve como um tampão de bloqueio. </li><li> Usando televisão de ponta pinça, remova cada membrana para ser usado por entre as folhas de protecção e coloque em um poço do 4-Bem prato Multi-. O número da matriz deve estar voltado para cima. </li><li> Em contacto com uma matriz de tampão, o corante azul das manchas desaparecerá, mas os anticorpos de captura são mantidas nos seus locais específicos. </li><li> Incubar durante 1 hora numa plataforma de agitação. Oriente a bandeja de modo que cada matriz final rochas para terminar em seu bem. A superfície da matriz deve ser completamente molhada e cobertos por tampão de bloqueio. </li><li> Enquanto as membranas são bloquear, dilui-se a quantidade desejada de lisado celular para um volume final de 1,5 ml com tampão de matriz 1. </li> &lt;li&gt; Tampão matriz Aspirar um dos poços da placa de 4 Bem-Multi e adicionar soluções diluídas lisado. Colocar a tampa no prato 4-Multi-Well. </li><li> Incubar durante a noite a 2-8 ° C numa plataforma oscilante. Um tempo de incubação mais curto pode ser utilizado se a sensibilidade óptima não é necessária. </li><li> Cuidadosamente remover cada membrana a partir da 4-Well prato múltipla e colocar nos recipientes individuais de plástico com 20 ml de tampão de lavagem 1X. O 4-Well prato múltipla não pode ser usada para completar os passos de lavagem. Lavar o 4-Bem-prato Multi com água deionizada ou destilada e seque bem. </li><li> Lavar cada membrana com 1X tampão de lavagem durante 10 minutos num agitador de plataforma oscilante. Repita duas vezes para um total de 3 lavagens. </li><li> Diluir o anti-fosfo-tirosina-peroxidase de rábano tampão de detecção (HRP) em 1X tampão de matriz 2, utilizando o factor de diluição, no rótulo do frasco. Pipetar 2,0 ml de uma solução diluída para cada poço do prato de 4 Multi-Well. </li><li> Remova cuidadosamente cada membrana de seu recipiente de lavagem.Permitir tampão em excesso para drenar a partir da membrana e voltar a membrana para o 4-Well prato Multi-diluído contendo o anti-fosfo-tirosina-HRP. Cobrir os poços com tampa. </li><li> Incubar durante 2 horas à temperatura ambiente numa plataforma oscilante. </li><li> Lavar cada matriz, tal como descrito anteriormente. </li><li> Conclua as etapas restantes, sem interrupção. Remova cuidadosamente cada membrana de seu recipiente de lavagem. Permitir tampão para drenar o excesso da membrana por blotting da aresta inferior em papel absorvente. Colocar cada membrana em uma folha de plástico protector, com o número de identificação para cima. </li><li> Pipeta de 1 ml do reagente preparado Chemi Misturar uniformemente para cada membrana. Utilizando menos do que 1 ml de Reagente Mix Chemi por membrana pode resultar na cobertura incompleta da membrana. A substituição de alguns reagentes de alta intensidade quimioluminescentes para Chemi Reagentes 1 e 2 podem causar um fundo aumentado ou diminuído, dependendo do sinal do reagente. </li><li> Cuidadosamente cubra com um plásticoprotetor da folha. Gentilmente suavizar as bolhas de ar e garantir Chemi Mix Reagente é distribuído uniformemente a todos os cantos de cada membrana. Incubar durante 1 min. </li><li> Toalhas de papel de posição em cima e dos lados do protetor da folha de plástico contendo as membranas e cuidadosamente esprema o excesso de reagente Mix Chemi. </li><li> Retire o protetor da folha superior de plástico e cuidadosamente colocar um laboratório absorvente sobre o topo das membranas para apagar de desconto em qualquer misturar reagentes restantes Chemi. </li><li> Deixando de membranas no protetor inferior da folha de plástico, cobrir as membranas com filme plástico com cuidado para delicadamente eliminar possíveis bolhas de ar. Envolver o envoltório excesso de plástico em torno da parte de trás da folha de protector a fim de que as membranas e protetor folha está completamente enrolada. </li><li> Coloque as membranas com os números de identificação voltadas para cima, de uma cassete de película auto-radiografia que não é utilizado com detecção de isótopos radioactivos. </li><li> Expor a película Kodak Luz BioMax para min 1-10. Várias exposições são Recomterminou. Alternativamente, as imagens podem ser coletados por meio de um gerador de imagens de quimiluminescência compatível, como a Carestream Health Imagem Estação 4000mm PRO. </li></ol> 3. Análise de Dados <ol><li> Sinais positivos observados em filme desenvolvido podem ser rapidamente identificados através da colocação da camada de transparência sobre a imagem da matriz e alinhando-a com os pares de pontos de referência impressos nos cantos para esta finalidade. O número de identificação estampado sobre a matriz deve ser colocado no lado esquerdo tinha. A localização dos controles e anticorpos de captura está listada no apêndice da folha de dados matriz humana Phospho RTK. </li><li> Densidades de pixel em filme desenvolvido pode ser coletado usando um scanner de transmissão modo como o Epson Perfection V750 PRO. </li><li> Criar um modelo para analisar a densidade de pixels em cada ponto da matriz utilizando um adequado programa de imaginação software de análise. Programas compatíveis incluem software ImageQuant ou ArrayVision da GE, Western sof Visãotware com modelos específicos para proteoma Matrizes Profiler, BioRad Quantidade One, Adobe Photoshop, NIH ImageJ, e Alphaview proteína simples. </li><li> Exportar valores de sinal para um arquivo de planilha para a manipulação de um programa como o Microsoft Excel. </li><li> Determinar o sinal de média (densidade de pixels) do par de pontos duplicados representam cada RTK. </li><li> Subtrair um sinal de fundo em média a partir de cada ponto. Usar um sinal a partir de uma área livre de matriz ou pontos de controlo negativo como valor de fundo. </li><li> Comparar os sinais correspondentes em diferentes matrizes para determinar a variação relativa dos níveis de fosforilação de RTK entre as amostras. </li></ol> 4. Resultados representativos Este protocolo demonstra como a matriz Phospho RTK-humano pode ser utilizado para rastrear os efeitos da estimulação do ligando e inibidores da fosforilação RTK. Na Figura 1, os dados são apresentados para MDA-MB-453 de células que foram tratados, tratados com 100 ng/ Ml rhNRG-β1/HRG-β1 (HRG-β1), durante 5 minutos, ou pré-tratados com inibidores conhecidos da família ErbB 8-11 antes HRGβ1 tratamento. Para as experiências de inibidor, as células foram incubadas com 1 uM HDS 029, 200 nM de PD 153035 ou PD 158780 200 nM em SFM durante três horas. Cada matriz foi incubado com 100 | ig de lisado. Um aumento da fosforilação é observado para ErbB2, ErbB3, ErbB4 e pontos de captura quando comparando células não tratadas com HRG-β1 tratado MDA-MB-453 células. O tratamento das células com os três inibidores da família ErbB selectivas antes da HRG-β1 incubação causou reduções na ErbB2, ErbB3, ErbB4 e fosforilação. Na Figura 2, os dados são apresentados para as células Kato III conhecidos para superexpressar a RTK R2 FGF. Neste conjunto de experiências, as células Kato III foram tratados, tratados com 100 ng / ml rhFGF ácido (FGF) e heparina 1 ug / ml, ou pré-tratados com inibidores selectivos R FGF 12-15 antes do tratamento comFGF e heparina. O inibidores PD173074, PD 161570, PD 166285 e foram utilizados a uma concentração de 1 uM e foram incubadas com as células Kato III para 3 horas em meio isento de soro. Embora não haja a fosforilação constitutiva de ambos EGF R e R2 FGF em células não tratadas KATO III, um aumento na fosforilação do FGF R2 foi observada após tratamento com FGF. Incubar as células com PD 173074, PD 161570, PD 166285 ou resultou em diminuição FGF e EGF R2 fosforilação R. Embora esses inibidores são conhecidos afectar a fosforilação dos membros da família FGF R, estes resultados demonstram a utilidade da matriz Phospho RTK-humano para monitorizar o efeito de uma concentração específica de inibidor pode ter sobre outros RTKs, tais como EGF R neste caso. <img alt="Figura 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/4199/4199fig1highres.jpg" src="/files/ftp_upload/4199/4199fig1.jpg" /> Figura 1. Indução e inibição da recepfosforilação da cinase da tirosina tor em células de câncer de mama. imagens matriz de Proteome Profiler Humanos Phospho RTK-matrizes e os perfis de histograma correspondentes são mostrados. MDA-MB-453 de células não tratadas ou foram tratadas com inibidores de RTK, seguido por tratamento com NRG-β1/HRG-β1. A matriz foi utilizada para monitorar os efeitos de inibidores da quinase na fosforilação em MDA-MB-453 células. <a href="http://www.jove.com/files/ftp_upload/4199/4199fig1large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img alt="Figura 2" fo:content-width="4.5in" fo:src="/files/ftp_upload/4199/4199fig2highres.jpg" src="/files/ftp_upload/4199/4199fig2.jpg" /> Figura 2. Imagens de matriz de indução e de inibição da fosforilação do receptor de tirosina-quinase em células de cancro gástrico. Partir de uma matriz de perfis Proteome Phospho RTK-humano e os perfis correspondentes de histograma são mostrados. Células Kato III foram untreated ou tratados com inibidores de RTK, seguido de tratamento com ácido FGF e heparina. <a href="http://www.jove.com/files/ftp_upload/4199/4199fig2large.jpg" target="_blank">para ver figura maior</a> . Referências <ol><li> Henriksson, ML, Edin, S., Dahlin, AM, Oldenborg, PA, Oberg, A., Van Guelpen, B., Rutegard, J., Stenling, R., &amp; Palmqvist, as células cancerosas R. colorretais ativar os fibroblastos adjacentes resultantes em FGF1/FGFR3 sinalização e invasão aumentada. Am. J. Pathol. 178, 1387-1394 (2011). </li><li> Morishita, A., Gong, J., Nomura, T., Yoshida, H., Izuishi, K., Suzuki, Y., Kushida, Y., Haba, R., D&#39;Armiento, J., &amp; Masaki, T. A utilização de proteínas de matriz para identificar receptores cinases de tirosina direccionáveis ​​para o tratamento de cancro do cólon humano. Int. J. Oncol. 37, 829-835 (2010). </li><li> Agaram, NP, Laquaglia, MP, Ustun, B., Guo, T. Wong, GC, Socci, ND, Maki, RG,DeMatteo, RP, Besmer, P., &amp; Antonescu, caracterização CR Molecular pediátricos de tumores do estroma gastrointestinal. Clin. Câncer Res. 14, 3204-3215 (2008). </li><li> Stommel, JM, Kimmelman, AC, Ying, H., Nabioullin, R., Ponugoti, AH, Wiedemeyer, R., Stegh, AH, Bradner, JE, Ligon, KL, Brennan, C., Chin, L., &amp; DePinho, RA coativação de tirosina-quinases receptoras afecta a resposta das células tumorais para terapias específicas. Science. 318, 287-290 (2007). </li><li> Agarwal, S., Zerillo, C., Kolmakova, J., Christensen, JG, Harris, LN, Rimm, DL, DiGiovanna, MP, e Stern, DF Association of constitutivamente activada do receptor do factor de crescimento de hepatócitos (Met) com resistência a um inibidor EGFR/Her2 dupla em não-pequenas células do cancro do pulmão de células. Br. J. Câncer. 100, 941-949 (2009). </li><li> Dewaele, BM, Floris, G., Finalet-Ferreiro, J., Fletcher, CD, Coindre, JM, Guillou, L., Hogendoorn, PC, Wozniak, A., Vanspauwen, V., Schoffski, P., MARYNEN, P., Vandenbergh, P., Sciot, R., &amp; Debiec-Rychter, M. Co-activated PDGFRA e EGFR são potenciais alvos terapêuticos sarcoma intimal. Cancer Res. 70, 7304-7314 (2010). </li><li> Yoon, YK, Kim, HP, Han, SW, Hur, HS, Oh, DY, Im, SA, Bang, YJ, &amp; Kim, TY Combinação de EGFR e MEK1 / 2 inibidor mostra efeitos sinérgicos por EGFR/HER3-dependent suprimindo AKT ativação em células cancerosas gástricas. Mol. Ther câncer. 8, 2526-2536 (2009). </li><li> Fry, DW, Nelson, JM, Slintak, V., Keller, PR, Rewcastle, GW, Denny, WA, Zhou, H., &amp; Bridges, propriedades bioquímicas e AJ antiproliferativa de 4 - [Ar (Alq) ilamino] piridopirimidinas, uma classe nova de produtos químicos potentes e específicos do factor de crescimento epidérmico receptor inibidor da tirosina quinase. Biochem. Pharmacol. 54, 877-887 (1997). </li><li> Bos,M., Mendelsohn, J., Kim, YM, Albanell, J., Fry, DW, e Baselga, J. PD153035, um inibidor da tirosina quinase, impede a activação do receptor de crescimento epidérmico e factor de inibição do crescimento de células cancerosas em um número de receptores- forma dependente. Clin. Câncer Res. 3, 2099-2106 (1997). </li><li> Stoll, SW, Kansra, S., Peshick, S., Fry, DW, Leopold, WR, Wiesen, JF, Sibilia, M., Zhang, T., Werb, Z., Derynck, R., Wagner, EF, &amp; Neoplasia Elder, JT utilização diferencial e localização de quinases de tirosina receptores ErbB na pele em comparação com os queratinócitos normais e malignas.. 3, 339-350 (2001). </li><li> Klutchko, SR, Zhou, H., Winters, RT, Tran, TP, Pontes, AJ, Althau, IW, Amato, DM, Elliott, WL, Meade, MA, Roberts, BJ, Fry, DW, Gonzales, AJ, Harvey , PJ, Nelson, JM, Sherwood, V., Han, HK, Pace, G., smaill, JB, Denny, WA, e Showalter, inibidores da quinase de tirosina de HD. 19. 6-alknyamides de 4-anilinoquinazolinas e 4-anilinopyrido [3,4-d] pirimidinas como inibidores irreversíveis da família ErbB de receptores tirosina-quinase. J. Med. Chem. 49, 1475-1485 (2006). </li><li> Kunii, K., Davis, L., Gorenstein, J., Hatch, H., Yashiro, M., Di Bacco, A., Elbi, C., &amp; Lutterbach, B. FGFR2-amplificados linhas de células gástricas cancerosas necessitam de FGFR2 ErbB3 e sinalização para o crescimento e sobrevivência. Câncer Res. 68, 2340-2348 (2008). </li><li> Zhou, W., Hur, W. McDermott, U., Dutt, A., Xian, W., Ficarro, SB, Zhang, J., Silva, SV, Brugge, J., Meyerson, M., Settleman, J . &amp; Gray, NS Uma abordagem orientada-estrutura para a criação de inibidores FGFR covalentes. Chem. Biol. 17, 285-295 (2010). </li><li> Batley, BL, Doherty, AM, Hamby, JM, Lu, GH, Keller, P., Dahring, TK, Hwang, O., Crickard, K., &amp; Panek Inibição RL de FGF-1, a actividade de cinase de tirosina receptora de PD 161570 , uma proteína nova-Tirosina-quinase inibidor. Life Sei. 62 143-150 (1998). </li><li> Panek, RL, Lu GH, Klutchko, SR, Batley, BL, Darhing, TK, Hamby, JM, Hallak, H., Doherty, AM, e Keiser, JA In vitro caracterização farmacológica de PD 166285, um novo potente nanomolar e amplamente activo inibidor da proteína tirosina quinase. J. Pharmacol. Exp. Ther. 283, 1433-1444 (1997). </li></ol>

The Human Phospho-RTK é uma alternativa econômica e rápida de métodos tradicionais, tais como imunoprecipitação (IP) ocidental para mudanças de triagem na fosforilação de TKU. Usando este método, os níveis relativos de 42 fosfo-RTKs foram rastreados simultaneamente utilizando uma única amostra de MDA-MB-453 ou de lisado de células Kato III. Além disso, este ensaio matriz exigido 2,5 horas de hands-on do tempo, tornando este método muito mais eficaz do que o tempo de realização de múltiplas IP-Westerns. Ao empregar um método de detecção por quimioluminescência, nenhum equipamento especializado para além do que é normalmente utilizado para a recolha de dados foi necessário ocidentais. Arrays são sensíveis e podem ser utilizadas para comparar mudanças na fosforilação causadas por ambos ligando e tratamento inibidor. Este método também permite a avaliação da selectividade inibidor para fora do alvo RTKs. Acertos positivos podem ser ainda avaliados utilizando um ensaio quantitativo tal como um ELISA. Para efetivamente empregar matrizes para o rastreio de alteraçõesna fosforilação, algumas diretrizes fundamentais experimentais devem ser seguidas. Em primeiro lugar, as experiências devem incluir amostras de controlo adequadas. Por exemplo, nestas experiências o efeito de inibidores em MDA-MB-453 ou III Kato lisados ​​foram medidos em amostras de lisados ​​preparados no mesmo dia. Apenas os dados de matriz, que são recolhidos dentro da mesma experiência devem ser analisados ​​para minimizar variações de sinal, devido a diferenças no manuseamento das amostras, o tempo de incubação, a técnica de pipeta, ou a técnica de lavagem. Intensidades de sinal comparando entre dois analitos na mesma membrana não é recomendada, uma vez que os anticorpos de captura não foram seleccionadas para ter afinidades paralelas.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td><a href="http://www.rndsystems.com/product_results.aspx?k=ary001" target="_blank">Human Phospho-RTK Array Kit</a></td>
			<td>R&amp;D Sytems</td>
			<td>ARY001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Tocris</td>
			<td>1167</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>Tocris</td>
			<td>1190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MDA-MB-453 cells</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kato III cells</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td><a href="http://www.rndsystems.com/product_results.aspx?k=396-hb" target="_blank">Recombinant Human NRG1-beta 1/HRG1-beta 1 EGF Domain</a></td>
			<td>R&amp;D Systems</td>
			<td>396-HB-050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td><a href="http://www.rndsystems.com/product_results.aspx?k=232-fa" target="_blank">Recombinant Human FGF acidic (aa 16-155)</a></td>
			<td>R&amp;D Systems</td>
			<td>232-FA-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HDS 029</td>
			<td>Tocris</td>
			<td>2646</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 153035</td>
			<td>Tocris</td>
			<td>1037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 158780</td>
			<td>Tocris</td>
			<td>2615</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 173074</td>
			<td>Tocris</td>
			<td>3044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 161570</td>
			<td>Tocris</td>
			<td>3724</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 166285</td>
			<td>Tocris</td>
			<td>3785</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BCA assay</td>
			<td>Pierce</td>
			<td>23225</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate-Buffer Saline (PBS)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettes and pipette tips</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Deionized or distilled water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Flat-tipped tweezers</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rocking platform shaker</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plastic containers with the capacity to hold 50 ml</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>For washing arrays</td>
		</tr>
		<tr>
			<td>Plastic transparent sheet protector</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plastic wrap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Absorbent lab wipes</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Autoradiography cassette</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Film developer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kodak BioMax Light Film</td>
			<td>Carestream Health</td>
			<td>178 8207</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Film developer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Image Station 4000MM PRO</td>
			<td>Carestream Health</td>
			<td>&nbsp;</td>
			<td>Chemiluminescence imager compatible with collection of western data may be used instead of film and a film developer.</td>
		</tr>
		<tr>
			<td>Epson Perfection Scanner</td>
			<td>Epson</td>
			<td>V750 PRO</td>
			<td>Flatbed scanner with transparency adaptor capable of transmission mode</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>GE,WesternVision, BioRad, Adobe, NIH, Protein Simple, etc</td>
			<td>&nbsp;</td>
			<td>ImageQuant, ArrayVision, Western Vision, Quantity One, Photoshop, ImageJ, AlphaView, or other compatible program</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Computer capable of running image analysis software and Microsoft Excel.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

profiling changes in receptor tyrosine kinase phosphorylation using antibody arrays - advertisement

A desregulação da expressão de receptores de tirosina-quinase (RTK) e fosforilação está frequentemente associada com a propagação e o desenvolvimento metastático de células cancerosas. Esforço significativo 1-3 tem sido focada em desenvolver inibidores específicos para alvejar RTKs e interromper as vias de sinalização aberrantes associados com estados de doença . 4-7 O objetivo deste estudo foi avaliar os efeitos de um seleto número de inibidores de fosforilação de TKU. Este estudo utilizou a matriz Phospho-RTK humanos, que é uma membrana de imunoensaio em sanduíche com base para monitorar aumentos ou diminuições na fosforilação de RTKs numerosas simultaneamente numa única amostra. Neste ensaio, as duas RTKs fosforiladas e não fosforilada presente em uma amostra de lisado são capturados por anticorpos discretos impressos em duplicado através de uma membrana de nitrocelulose, o tamanho de uma lâmina de microscópio. Após lavagem, as matrizes são incubadas com anti-fosfo-tirosina-peroxidase de rábano (HRP),que sanduíches com TKUs fosforilados capturados na matriz. Após um passo de lavagem em segundo lugar, as matrizes são incubados com reagentes quimioluminescentes. Sinal gerado em cada ponto da matriz é proporcional à quantidade de proteína fosfo-ligado por cada anticorpo de captura. Nestas experiências, as matrizes foram utilizadas para medir o aumento de fosforilação após a estimulação do ligando ou do cancro da mama MDA-MB-453 ou III KATO células de carcinoma gástrico, bem como para caracterizar diminuições na fosforilação associados com o tratamento de células com inibidores selectivos para ErbB ou família FGF membros R.

Matrizes de anticorpos proteoma Profiler são uma forma conveniente e eficiente para a tela para mudanças no receptor tirosina quinase (RTK) fosforilação sem a realização de numerosos imunoprecipitação (IP) Westerns. A matriz RTK ARY001 Human permite a medição qualitativa das RTK múltiplos numa única amostra usando detecção por quimioluminescência.

Alterações de perfil em Receptor Fosforilação da tirosina quinase utilizando matrizes de anticorpos - Publicidade

The dysregulation of receptor tyrosine kinase (RTK) expression and phosphorylation is frequently associated with the development and metastatic spread of ...

profiling-changes-receptor-tyrosine-kinase-phosphorylation-using

Without Section

Profiling Changes in Receptor Tyrosine Kinase Phosphorylation using Antibody Arrays - ADVERTISEMENT

11.5K visualizações.  R&D Systems, Inc.. Matrizes de anticorpos proteoma Profiler são uma forma conveniente e eficiente para a tela para mudanças no receptor tirosina quinase (RTK) fosforilação sem a realização de numerosos imunoprecipitação (IP) Westerns. A matriz RTK ARY001 Human permite a medição qualitativa das RTK múltiplos numa única amostra usando detecção por quimioluminescência.

Vídeo: Alterações de perfil em Receptor Fosforilação da tirosina quinase utilizando matrizes de anticorpos - Publicidade

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Monitoramento adsorção de proteínas com Solid-state Nanoporos

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Research

JoVE Journal

Bioengenharia

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Determinação da atividade fagocítica de amostras de anticorpos Clínica

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Imunologia e Infecção

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Quantitativo de alta capacidade ensaio de citotoxicidade de célula única para as células T.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Quantificação automatizada de fluorescência em Synaptic<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Neurociência

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Perfil de anticorpos por sistemas de imunoprecipitação luciferase (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Biologia

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western Blotting: Preparação de amostras para detecção

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood 1,2,3. Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) 1,2 and precocious puberty 4. Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR.
Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well.
The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies 1,4,5,6,7,8. As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation 4 as well as to alter the rate of degradation of KISS1R 9. Interestingly, our studies indicate that KISS1R is degraded by the proteasome, as opposed to the classic lysosomal degradation described for most G protein-coupled receptors 9. In the example presented here, degradation of the KISS1R is investigated in Human Embryonic Kidney Cells (HEK-293) transiently expressing Myc-tagged KISS1R (MycKISS1R) and treated with proteasome or lysosome inhibitors. Cell lysates are immunoprecipitated using an agarose-conjugated anti-myc antibody followed by western blot analysis. Detection and quantification of MycKISS1R on blots is performed using the LI-COR Odyssey Infrared System. This approach may be useful in the study of the degradation of other proteins of interest as well.

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

Mutagênese e análise de mutações genéticas nos receptores de Seqüência GC-rica KISS1 identificados em humanos com distúrbios reprodutivos

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Atividades de monitoramento quinases e fosfatases através do ciclo celular por raciométrica FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Doseamento do atividade da quinase de LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Concepção e utilização de matrizes quemostato Multiplexados

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...