Este trabajo fue financiado por R &amp; D Systems, Inc.

1. Preparación de la muestra <ol><li> Recoger lisados ​​de tratamiento, tratamiento ligando, o un inhibidor de las células y ligandos tratados siguiendo las directrices de la hoja de datos array Humanos Phospho-RTK. El tampón de lisis que ha sido optimizado para este kit. La sustitución de otros tampones de lisis puede afectar al rendimiento final de la matriz. Para evitar la degradación proteolítica de la muestra, añadir 10 g / ml aprotinina, 10 mg / ml de leupeptina, y 10 ug / ml de pepstatina A para el volumen de tampón de lisis que se requiere para cada preparación de lisado celular fresca para cada uso. </li><li> Concentración lisado se medirá usando un ácido bicinconínico (BCA) de ensayo. Concentración de la muestra se puede ajustar empíricamente para una sensibilidad óptima y bajo fondo. Una gama de 100-300 g de lisado se recomienda como un punto de partida inicial. </li><li> Ciclos descongelación / congelación de las muestras deben ser evitados y almacenamiento adecuado de los lisados ​​se requiere para un rendimiento óptimo. Descongele las muestras de lisado en hielo. </li></ol><ol><li> Lleve todos los componentes del kit a temperatura ambiente antes de su uso. Para evitar la contaminación, use guantes al realizar los procedimientos. </li><li> Pipetear 2,0 ml de búfer de matriz 1 en cada pocillo de la 4-Bien Múltiples plato para ser utilizado. Buffer matriz 1 sirve como un amortiguador bloque. </li><li> Utilizando pinzas de punta plana, retire las membranas a utilizar de entre las hojas protectoras y colocar en una fuente de la 4-Bueno multi-plato. El número de serie debe estar mirando hacia arriba. </li><li> Al entrar en contacto con el tampón de matriz 1, el colorante azul de las manchas desaparecerán, pero los anticuerpos de captura son retenidos en sus ubicaciones específicas. </li><li> Incubar durante 1 h en una plataforma oscilante. Oriente la bandeja de manera que cada extremo matriz rocas para terminar en su bien. La superficie de la matriz debe ser completamente mojados y cubiertos por tampón de bloqueo. </li><li> Mientras que las membranas están bloqueando, se diluye la cantidad deseada de lisado celular a un volumen final de 1,5 ml con tampón de matriz 1. </li> &lt;li&gt; Buffer matriz Aspirar una de las fuentes de la 4-Bueno multi-plato y añadir soluciones diluidas lisado. Coloque la tapa sobre el 4-Bueno multi-plato. </li><li> Incubar durante la noche a 2-8 ° C en una plataforma oscilante. Un tiempo de incubación más corto puede ser usado si una sensibilidad óptima, no es necesario. </li><li> Retire cuidadosamente cada membrana de la 4-Bien Múltiples plato y colocar en recipientes individuales de plástico con 20 ml de tampón de lavado 1X. El 4-Bueno Múltiples plato no puede ser utilizado para completar los pasos de lavado. Enjuague el 4-Bueno multi-plato con agua desionizada o destilada y secar bien. </li><li> Lavar cada membrana con tampón de lavado 1X durante 10 min en un agitador de plataforma oscilante. Repetir dos veces para un total de 3 lavados. </li><li> Diluir el anti-fosfo-tirosina-peroxidasa de rábano (HRP) en tampón de detección búfer de matriz 1X 2 usando el factor de dilución en la etiqueta del vial. Pipetear 2,0 ml de solución diluida en cada pocillo de la 4-Bien multi-plato. </li><li> Retire con cuidado cada membrana de su recipiente de lavado.Permitir que el exceso de tampón para drenar de la membrana y la membrana volver a la 4-Bien Múltiples plato que contiene la solución diluida de anti-fosfo-tirosina-HRP. Cubrir los pocillos con la tapa. </li><li> Incubar durante 2 horas a temperatura ambiente en una plataforma oscilante. </li><li> Lavar cada matriz como se ha descrito anteriormente. </li><li> Complete los pasos restantes sin interrupción. Retire con cuidado cada membrana de su recipiente de lavado. Permitir que el exceso de tampón para drenar de la membrana por transferencia el borde inferior sobre el papel absorbente. Colocar cada membrana en un protector de lámina de plástico con el número de identificación hacia arriba. </li><li> Pipetear 1 ml del reactivo preparado Chemi Mezclar uniformemente sobre cada membrana. Utilizar menos de 1 ml de mezcla de reactivos Chemi por membrana puede dar lugar a una cobertura incompleta de membrana. La sustitución de algunos reactivos de alta intensidad quimioluminiscente para Chemi Reactivos 1 y 2, pueden provocar ya sea aumentado o fondo señal disminuida dependiendo del reactivo. </li><li> Con cuidado, cubrir con un plásticolámina protectora. Suavemente suavizar las burbujas de aire y asegurar Mix Chemi reactivo se distribuye uniformemente a todos los rincones de cada membrana. Incubar durante 1 min. </li><li> Toallas de papel en la posición superior y los lados del protector de lámina de plástico que contiene las membranas y cuidadosamente exprimir el exceso de reactivo Mix Chemi. </li><li> Retire el protector superior lámina de plástico y coloque cuidadosamente un laboratorio paño absorbente en la parte superior de las membranas para borrar cualquier resto de mezcla de reactivos Chemi. </li><li> Dejando de membranas en el protector de plástico de la hoja inferior, cubra las membranas con un envoltorio de plástico con cuidado de alisar suavemente las burbujas de aire. Envolver el exceso de envoltura de plástico alrededor de la parte posterior del protector de la hoja de modo que las membranas y el protector de hojas están completamente envuelto. </li><li> Coloque las membranas con los números de identificación hacia arriba, en un casete de película de autorradiografía que no se utiliza con la detección de isótopos radiactivos. </li><li> Exponer a la película Kodak BioMax Light por 1-10 min. Exposiciones múltiples son recomterminado. Alternativamente, las imágenes pueden ser recolectados a través de una cámara de quimioluminiscencia compatible, como el de Carestream Health Image Station 4000MM PRO. </li></ol> 3. Análisis de Datos <ol><li> Señales positivas observadas en la película revelada se pueden identificar rápidamente mediante la colocación de la superposición de la transparencia en la imagen matriz y alinearlo con los pares de puntos de referencia impresas en las esquinas para este propósito. El número de identificación estampada en la matriz debe ser colocado en el lado izquierdo tenía. La ubicación de los controles y los anticuerpos de captura se enumeran en el Apéndice de la hoja de datos array Humanos Phospho-RTK. </li><li> Densidades de píxeles en la película desarrollada puede ser recogida mediante un escáner modo de transmisión, tales como el Epson Perfection V750 PRO. </li><li> Crear una plantilla para analizar la densidad de píxeles en cada punto de la matriz mediante un adecuado análisis de imágenes de software. Programas compatibles incluyen software ImageQuant o ArrayVision de GE, sof Western Visiontware con plantillas específicas para proteoma Arrays Profiler, BioRad Cantidad One, Adobe Photoshop, ImageJ NIH, y AlphaView proteína simple. </li><li> Exportar valores de señal en un archivo de hoja de cálculo para su manipulación en un programa como Microsoft Excel. </li><li> Determinar la señal promedio (densidad de píxeles) del par de puntos duplicados que representan cada RTK. </li><li> Resta una señal de fondo promedio de cada lugar. Utilizar una señal de un área clara de la matriz o de puntos de control negativo como un valor de fondo. </li><li> Comparar las señales correspondientes en diferentes matrices para determinar el cambio relativo en los niveles de fosforilación de RTK entre las muestras. </li></ol> 4. Los resultados representativos Este protocolo se muestra cómo el humano Phospho-RTK matriz puede utilizarse para cribar los efectos de la estimulación del ligando y los inhibidores de la fosforilación de RTK. En la Figura 1, se muestran datos para MDA-MB-453 células que no fueron tratadas, se trató con 100 ng/ Ml rhNRG-β1/HRG-β1 (HRG-β1) durante 5 min, o pre-tratados con inhibidores conocidos de la familia ErbB 8-11 antes de HRGβ1 tratamiento. Para los experimentos de inhibidor, se incubaron las células con 1 mM de HDS 029, 200 nM EP 153035, EP 158780 o 200 nM en SFM durante tres horas. Cada matriz se incubó con 100 g de lisado. Un incremento en la fosforilación se observa para ErbB2, ErbB3, y ErbB4 puntos de captura cuando se comparan las células no tratadas con HRG-β1 tratados MDA-MB-453 células. El tratamiento de las células con los tres inhibidores de la familia erbB selectivos antes de HRG-β1 incubación causó reducciones en ErbB2, ErbB3, ErbB4 y la fosforilación. En la Figura 2, se muestran datos para las células Kato III conocidos para sobreexpresar la R2 RTK FGF. En este conjunto de experimentos, las células Kato III se dejaron sin tratar, se trataron con 100 ng / ml rhFGF ácido (FGF) y heparina 1 g / ml, o pretratadas con FGF R inhibidores selectivos de 12-15 antes del tratamiento conFGF y la heparina. El PD173074 inhibidores, PD 161570, y PD 166285 se utilizaron a una concentración de 1 mM y se incubaron con las células Kato III durante 3 horas en medio libre de suero. Si bien no es la fosforilación constitutiva de ambos EGF-R y R2 FGF en células no tratadas KATO III, un incremento en la fosforilación R2 FGF se observó a tratamiento FGF. La incubación de las células con la EP 173074, EP 161570, EP 166285 o como resultado una disminución FGF R2 y la fosforilación de EGF-R. Aunque estos inhibidores son conocidos afecta a la fosforilación de los miembros de la familia de FGF R, estos resultados demuestran la utilidad de la Human Phospho-RTK matriz para monitorizar el efecto de una concentración específica de inhibidor se puede tener en RTKs otros, tales como EGF-R en este 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. La inducción y la inhibición de receptorestor fosforilación de la tirosina cinasa en las células del cáncer de mama. imágenes de matriz de Proteome Profiler Humanos RTK Phospho-arrays y los perfiles correspondientes histogramas se muestran. MDA-MB-453 células se dejaron sin tratar o tratados con inhibidores de RTK seguido de tratamiento con NRG-β1/HRG-β1. La matriz se utilizó para monitorizar los efectos de los inhibidores de la fosforilación quinasa en MDA-MB-453 células. <a href="http://www.jove.com/files/ftp_upload/4199/4199fig1large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</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. Inducción y la inhibición de la fosforilación de tirosina quinasa del receptor en células de cáncer gástrico. Imágenes matriz a partir de un proteoma humano Profiler Phospho-RTK matriz y los perfiles de histograma correspondiente se muestran. Kato células III fueron untreado o tratados con inhibidores de la RTK seguido de tratamiento con FGF ácido y heparina. <a href="http://www.jove.com/files/ftp_upload/4199/4199fig2large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . Referencias <ol><li> Henriksson, ML, Edin, S., Dahlin, AM, Oldenborg, PA, Oberg, A., Guelpen Van, B., Rutegard, J., Stenling, R., &amp; Palmqvist, R. células del cáncer colorrectal activan los fibroblastos adyacentes resultantes en FGF1/FGFR3 señalización y la invasión creciente. Soy. 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. El uso de la matriz de proteína para identificar los receptores de tirosina quinasas dirigibles para el tratamiento de cáncer de colon 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, CR Caracterización molecular de los tumores pediátricos del estroma gastrointestinal. Clin. Cancer 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 coactivación de receptores de tirosina quinasas afecta a la respuesta de las células tumorales a las terapias dirigidas. Science. 318, 287-290 (2007). </li><li> Agarwal, S., Zerillo, C., Kolmakova, J., Christensen, JG, Harris, LN, Rimm, DL, DiGiovanna, MP, y Stern, DF Asociación de constitutivamente activada del receptor de factor de crecimiento de hepatocitos (Met) con resistencia a un inhibidor dual EGFR/Her2 en la no-células pequeñas células de cáncer de pulmón. fr. J. Cancer. 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-PDGFRA y activado EGFR son objetivos terapéuticos potenciales en el sarcoma intimal. Cancer Res. 70, 7.304 a 7.314 (2010). </li><li> Yoon, YK, Kim, HP, Han, SW, Hur, HS, Oh, DY, Im, SA, Bang, YJ, y Kim, Combinación IDAD del EGFR y MEK 1/2 inhibidor muestra efectos sinérgicos al suprimir EGFR/HER3-dependent AKT activación en células humanas de cáncer gástrico. Mol. Ther cáncer. 8, 2526-2536 (2009). </li><li> Fry, DW, Nelson, JM, Slintak, V., Keller, PR, Rewcastle, GW, Denny, WA, Zhou, H., &amp; Bridges, propiedades bioquímicas y AJ antiproliferativos de 4 - [Ar (ALK) ilamino] piridopirimidinas, una nueva clase química de potente y específico inhibidor de factor de crecimiento epidérmico receptor tirosina quinasa. Biochem. Pharmacol. 54, 877-887 (1997). </li><li> Bos,M., Mendelsohn, J., Kim, YM, Albanell, J., Fry, DW, y Baselga, J. PD153035, un inhibidor de tirosina quinasa, evita que el factor de crecimiento epidérmico activación del receptor e inhibe el crecimiento de células cancerosas en un número de receptores- forma dependiente. Clin. Cancer 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, y personas mayores, la utilización de JT diferencial y la localización de tirosina cinasas receptoras erbB en la piel en comparación con los queratinocitos normales y malignas. Neoplasia. 3, 339-350 (2001). </li><li> Klutchko, SR, Zhou, H. Winters, RT, Tran, TP, Bridges, 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, y Showalter, HD inhibidores de la tirosina cinasa. 19. 6-alknyamides de 4-anilinoquinazolinas y 4-anilinopyrido [3,4-d] pirimidinas como inhibidores irreversibles de la familia ErbB de receptores tirosina quinasa. 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-amplificación de líneas celulares de cáncer gástrico requieren FGFR2 y ErbB3 de señalización para el crecimiento y la supervivencia. Cancer Res. 68, 2340-2348 (2008). </li><li> Zhou, W., Hur, W. McDermott, U., Dutt, A., Xian, W., Ficarro, SB, Zhang, J., Sharma, SV, Brugge, J., Meyerson, M., Settleman, J . &amp; Gray, NS Un enfoque estructura guiada a la creación de inhibidores covalentes FGFR. 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 RL La inhibición de la actividad de FGF-1 receptor de la tirosina cinasa de PD 161570 , una proteína nuevaDe la tirosina quinasa. Life Sci. 62 143-150 (1998). </li><li> Panek, RL, Lu GH, Klutchko, SR, Batley, BL, Darhing, TK, Hamby, JM, Hallak, H., Doherty, AM, y Keiser, JA En la caracterización farmacológica in vitro de la EP 166285, una nueva y potente nanomolar y ampliamente tirosina activo inhibidor de la proteína quinasa. J. Pharmacol. Exp. Ther. 283, 1433-1444 (1997). </li></ol>

The Human fosfo-RTK es una alternativa económica y rápida a los métodos tradicionales, como la inmunoprecipitación (IP) Western detección de cambios en la fosforilación de RTK. Usando este método, los niveles relativos de 42 fosfo-RTK se rastrearon simultáneamente usando una sola muestra de MDA-MB-453 o lisado celular Kato III. Además, este ensayo requiere array 2,5 horas de práctica en el tiempo, por lo que este método mucho más efectivo que el tiempo de realización de múltiples IP-Oeste. Mediante el empleo de un método de detección de quimioluminiscencia, ningún equipo especializado más allá de lo que se suele utilizar para recoger datos occidentales se requería. Las matrices son sensibles y pueden ser utilizados para comparar los cambios en la fosforilación causada tanto por ligando y el tratamiento con inhibidor. Este método también permite la evaluación de la selectividad de inhibidor de RTK fuera de objetivo. Éxitos positivos pueden ser adicionalmente evaluada usando un ensayo cuantitativo tal como un ELISA. Para emplear eficazmente las matrices para la detección de cambiosen la fosforilación, algunas pautas experimentales clave deben ser seguidas. En primer lugar, los experimentos deben incluir muestras de control adecuadas. Por ejemplo, en estos experimentos el efecto de los inhibidores de MDA-MB-453 o Kato III lisados ​​se midió en las muestras de lisado preparadas el mismo día. Sólo los datos de matriz que se recoge dentro del mismo experimento deben ser analizados para minimizar las variaciones en la señal debido a las diferencias en el manejo de muestras, tiempo de incubación, la técnica de pipeta, o la técnica de lavado. Comparación de las intensidades de señal entre dos analitos en la misma membrana no es recomendable, ya que los anticuerpos de captura no fueron seleccionados para tener 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

La desregulación de la tirosina quinasa del receptor (RTK) de expresión y fosforilación se asocia frecuentemente con el desarrollo y la propagación metastásica de las células cancerosas. Esfuerzo significativo 1-3 se ha centrado en desarrollar inhibidores para orientar las RTK específicas e interrumpir las rutas de señalización aberrantes asociados con estados de enfermedad 4-7. El propósito de este estudio fue medir los efectos de un número selecto de los inhibidores de la fosforilación RTK. Este estudio utilizó el Humano Phospho-RTK matriz, que es un inmunoensayo de sándwich basado en membrana para controlar los aumentos o disminuciones en la fosforilación de RTK numerosos simultáneamente en una única muestra. En este ensayo, tanto las RTK fosforilados y no fosforilados presentes en una muestra de lisado son capturados por los anticuerpos discretas impresas por duplicado a través de una membrana de nitrocelulosa del tamaño de un portaobjetos de microscopio. Después del lavado, los arrays se incubaron con anti-fosfo-tirosina-peroxidasa de rábano (HRP),que sándwiches con RTK fosforilados capturados en la matriz. Después de una segunda etapa de lavado, los arrays se incubaron con reactivos quimioluminiscentes. Señal generada en cada punto de matriz es proporcional a la cantidad de fosfo-proteína unida por cada anticuerpo de captura. En estos experimentos, las matrices se utilizan para medir el aumento de la fosforilación después de la estimulación del ligando de cualquiera de cáncer de mama MDA-MB-453 o células KATO III de carcinoma gástrico, así como para caracterizar la disminución en la fosforilación asociados con el tratamiento de las células con inhibidores selectivos hacia ErbB o FGF R familiares.

Proteome arrays de anticuerpos Profiler son una manera eficiente y costo conveniente para la detección de cambios en receptores tirosina quinasa (RTK) fosforilación sin realizar numerosas inmunoprecipitación (IP) Westerns. La matriz ARY001 RTK humano permite la medición cualitativa de las RTK múltiples en una única muestra usando la detección de quimioluminiscencia.

Los cambios de perfiles en la fosforilación de la tirosina quinasa del receptor del uso de arrays de anticuerpos - Publicidad

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 vistas.  R&D Systems, Inc.. Proteome arrays de anticuerpos Profiler son una manera eficiente y costo conveniente para la detección de cambios en receptores tirosina quinasa (RTK) fosforilación sin realizar numerosas inmunoprecipitación (IP) Westerns. La matriz ARY001 RTK humano permite la medición cualitativa de las RTK múltiples en una única muestra usando la detección de quimioluminiscencia.

Video: Los cambios de perfiles en la fosforilación de la tirosina quinasa del receptor del uso de arrays de anticuerpos - Publicidad

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.

Monitoreo adsorción de proteínas de estado sólido 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 ...

Investigación

JoVE Journal

Bioingeniería

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.

La determinación de la actividad fagocítica de los anticuerpos de muestras clínicas

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

Inmunología e Infección

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.

Cuantitativo de alto rendimiento de una sola célula de ensayo de citotoxicidad de las 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.

La cuantificación automática de la fluorescencia en el 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 ...

Neurociencias

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.

Perfiles de anticuerpos por los sistemas de inmunoprecipitación luciferasa (LIPS)

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

Biología

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 Blot: Preparación de muestras para la detección

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.

Mutagénesis y análisis de mutaciones genéticas en la secuencia del receptor GC-ricos KISS1 identificado en el hombre con Trastornos del aparato reproductor

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.

Seguimiento de las actividades de quinasa y fosfatasa través del ciclo celular por Proporcional 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.

Ensayo de la actividad quinasa 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.

Diseño y uso de arrays de quimiostato multiplexados

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