Esta investigación fue apoyada por el Programa de Investigación Intramural del NIDCR.

1. Esquemática general: Este video muestra los pasos necesarios para realizar el ensayo LIPS. Antígenos se expresan en células COS1 como recombinante luciferasa Renilla (RUC) del antígeno-fusiones, y extractos crudos obtenidos y utilizados sin purificación. El ensayo LIPS se inicia mediante la incubación de porquería e-Ruc antígeno extractos con sueros de pacientes en pocillos de microtitulación. La mezcla de antígeno-anticuerpo se transfiere a una placa de 96 pozos de filtración que contienen la proteína A / G cuentas para capturar las moléculas de IgG. Después de lavar la placa de filtro que contiene la proteína A / anticuerpo G cuentas, con destino Ruc-antígeno se mide por la adición de sustrato coelenterazine y unidades de luz se miden con un luminómetro. PARTE 1: La transfección de plásmidos para la producción de proteínas de fusión antígeno-Renilla Set-up: COS1 células se cultivan en DMEM-FCS al 10% utilizando los protocolos estándar de cultivo de tejidos. Plásmidos de luciferasa Renilla fusiones se han descrito anteriormente 1. ADN de estos plásmidos se prepara utilizando un kit de Qiagen Midiprep. El rendimiento debe ser de aproximadamente 1 mg -3. Medir la concentración de ADN y almacena como un mg 1000 / ml de solución madre a -20 ° C. Procedimiento: <ol><li> Un día antes de la transfección, dividir células COS-1 en los nuevos 100 x 20 mm platos en aproximadamente 2 x 10 6 por placa y se incuba a 37 ° C. </li><li> Al día siguiente, la células COS-1 debe ser un 80-95% confluente. Etiqueta de 1,5 ml tubos de microcentrífuga de polipropileno para cada plásmido de ADN que se transfectadas. Permita que el reactivo de transfección FuGENE-6, que se conserva a 4 ° C, para calentar a temperatura ambiente. </li><li> Añadir 94 l de Opti-MEM medios de comunicación a cada tubo de microcentrífuga. A continuación añadimos 6 l de FuGENE 6 a los medios de Opti-MEM sin tocar la pared lateral. </li><li> Incubar la mezcla durante 5 minutos a temperatura ambiente. </li><li> Añadir 1.2 g (desde el almacén de ADN 1mg/ml) de plásmido para la fusión de antígeno Renilla luciferase construir. Mezcla y se incuba la mezcla durante 15 minutos a temperatura ambiente. </li><li> La transferencia de la solución de ADN-FuGENE 6 Opti-MEM a las células por goteo de manera uniforme en los medios de comunicación de las células COS1. </li></ol> PARTE 2: Recolección de antígeno Renilla-Fusions <ol><li> Dos días después de la transfección, las células COS-1 se cosechan. Este se inicia con la eliminación de los medios de comunicación y luego enjuagar las células con 6 ml de PBS. Después de la decantación de la PBS, la pipeta a la basura cualquier PBS residual de la placa de cultivo de tejidos. </li><li> Añadir 1,4 ml de solución amortiguadora de lisis frío compuesto por 50 mM Tris, pH 7,5, 100 mM NaCl, 5 mM MgCl 2, 1% Triton X-100, 50% de glicerol y los inhibidores de la proteasa (2 comprimidos de cóctel completo inhibidor miniprotease por 50 ml de tampón de lisis). Las células de la cosecha con un raspador de células y rápida transferencia de la mitad de la lisado de cada uno de dos tubos de 1,5 ml de microcentrífuga en hielo. </li><li> A Sonifier Branson 150 se utiliza para romper las celdas abiertas. Coloque el tubo de microcentrífuga que contiene el lisado celular en el hielo y el pulso durante 5 segundos, 5 segundos y 5 segundos con la configuración de ultrasonidos de 2, 2 y 4, respectivamente. </li><li> Centrifugar el lisado celular a 12.500 rpm durante dos vueltas 4 minutos a 4 ° C. Después de la primera vuelta, invertir suavemente los tubos para eliminar los restos poco adjunta de la pared lateral del tubo. Después de la segunda vuelta, transferir cuidadosamente el sobrenadante, sin alterar el sedimento, a partir de los dos tubos a un tubo de microcentrífuga nuevo en hielo. </li><li> Calcular las unidades de luz (UGM) por l de lisado. Para medir la LU, diluir 1 l de lisado con 8 l de PBS en un tubo de microcentrífuga nuevo. Directamente añadir 100 ml de sustrato coelenterazine 1X a la mezcla diluida y de inmediato medidas de luminiscencia en el tubo con un tubo luminómetro (20/20 n Turner Científico) con una lectura de 5 segundos. </li><li> Tienda el lisado Ruc-antígeno a -20 ° C durante 1-2 días o almacén para un período más largo de los tiempos en alícuotas a -80 ° C. </li></ol> PARTE 3: Preparación de una placa Sera Maestro <ol><li> Hacer una placa maestra sueros añadiendo primero 450 l de tampón A (50 mM Tris, pH 7,5, 100 mM NaCl, 5 mM MgCl 2, 1% Triton X-100) a cada pocillo de una placa de polipropileno de 96 pozos profundos de microtitulación . En este paso, un colorante rojo, fenol, también se pueden incluir en un buffer (concentración final es de 0,2 mg / ml en tampón A) para actuar como un marcador para el monitoreo futura incorporación de la muestra de suero y otras medidas del ensayo LIPS. </li><li> A continuación se añaden 50 l de suero de cada muestra a los diferentes pozos que contiene 450 l de tampón A. Nótese que esta es una dilución 1:10 de los sueros en buffer A. Por lo general los sueros no se agrega a los dos últimos pozos de la placa principal, ya esto está reservado para los espacios de amortiguación. </li><li> Antes de utilizar la placa principal, que es ampliamente sacudida (1-2 horas) en una plataforma de los rotadores. El suero de la placa principal es estableble de al menos 1 mes (o más) a 4 ° C, si se almacena correctamente para evitar la evaporación. </li><li> Como se describe más adelante, esta placa principal dispone de 10 l de suero diluido para ser eliminado debido a que en repetidas ocasiones el perfil de los sueros contra múltiples antígenos. Grandes y pequeñas placas maestras también pueden ser empleados. Sellar la placa con Parafilm y cuando no se está utilizando. </li></ol> PARTE 4: ensayo LIPS <ol><li> Polipropileno de 96 placas de microtitulación poco profundas y se utilizan para los sueros. En el primer paso, agregar 40 ml de un buffer a cada pocillo de la placa de 96 pocillos utilizando una micropipeta de 8 canales. </li><li> A continuación, tomar 10 l de suero diluido (1 l equivalente sueros) de la placa principal y añadir directamente a cada pocillo de la placa de trabajo utilizando una micropipeta de 8 canales. </li><li> Una mezcla maestra que contiene el extracto Ruc-antígeno está al lado de formularse de manera que 1 X 10 7 unidades de luz (LU) se añade en 50 l de tampón A a cada pocillo. Menos insumos (tan poco como 1 x 10 6) también se puede utilizar, pero el resultado en un menor rango dinámico. Hacer esta mezcla principal y pipeta de 50 l de mezcla de Ruc-antígeno en cada pocillo. </li><li> Se incuba la placa en un agitador rotatorio durante 1 hora a temperatura ambiente. </li><li> Durante la incubación, añadir 5 l de una suspensión del 30% de proteína Ultralink A / G cuentas (Pierce Biotecnología, Rockford, IL) en PBS en el fondo de cada pocillo de una nueva placa de filtro de 96 HTS (Millipore, Bedford, MA) . </li><li> Después de la incubación de 1 hora, la transferencia de los 100 l Ruc antígeno-anticuerpo mezcla de reacción a 96 y placas de filtro HTS que contienen la proteína A / G a través de cuentas de una micropipeta de 8 canales. </li><li> Se incuba la placa de 96 pozos de filtro en un agitador rotatorio durante 1 hora más a temperatura ambiente. </li><li> A continuación se lava la placa de filtro en un colector de vacío. Cada pocillo se lava 8 veces con 100 l de tampón A, seguido por dos veces con 100 l de PBS. Esto se puede realizar manualmente o con una estación de trabajo pipeteo robótica. </li><li> Tras el último lavado, el vacío está apagado. Retire la placa del filtro y séquelo con una pila de toallas de papel o papel de filtro y asegúrese de eliminar la humedad en la parte superior e inferior de la placa. </li></ol> PARTE 5: análisis de medición de luminiscencia y datos Set-up: A Berthold LB 960 Centro microplaca luminómetro se utiliza para determinar la luminosidad en cada pocillo utilizando un solo inyector. Una vez que la máquina está encendida, lavar los inyectores con agua destilada H 2 O con el ciclo de lavado del inyector. Coelenterazine sustrato es preparado con el kit de Promega Renilla sustrato como se describe por el fabricante. Generalmente de 6 ml de la mezcla de sustrato coelenterazine 1X (es decir, 60 acciones coelenterazine l más 6 ml de 1X buffer) se prepara para el cebado de la máquina en funcionamiento un plato lleno de 96 pozos. Antes de analizar la placa, el LB Berthold 960 Centro microplaca luminómetro se ceba con sustrato coelenterazine 1X. Abrir un archivo de programa que contiene la configuración de la inyección del sustrato y la lectura de la placa. En estas mediciones, 50 l de sustrato coelenterazine se inyecta, la placa se agita durante 2 segundos, seguido de un 5 seg de lectura de luminiscencia. <ol><li> Aunque el luminómetro placa tiene la capacidad de leer toda la placa, una lectura parcial de la placa también puede ser seleccionado (en el menú de lectura). Iniciar el programa, que inicia la lectura de la placa. </li><li> Después de correr, quitar la placa del filtro de microtitulación con prontitud para evitar derrames en el luminómetro. Exportar los datos generados con el programa MikroWin en un formato Excel para su análisis. </li><li> Se recomienda la evaluación de los sueros de dos carreras independientes. Los datos se promedian para generar los valores título de LU para cada muestra. Estos valores aún se puede ajustar para restar los espacios de amortiguación. </li><li> Análisis de datos adicionales, tales como la determinación de la corte se puede calcular tomando la media más 3 o 5 desviaciones estándar de los valores de control. </li></ol>

<ol>
	<li>Burbelo, P. D., Goldman, R., Mattson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+immunoprecipitation+method+for+quantitatively+measuring+antibody+responses+in+clinical+sera+samples+by+using+mammalian-produced+Renilla+luciferase-antigen+fusion+proteins.">A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.</a> BMC Biotechnol. 5, 22-22 (2005).</li><li>Burbelo, P. D., Leahy, H. P., Iadarola, M. J., Nutman, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Four-Antigen+Mixture+for+Rapid+Assessment+of+Onchocerca+volvulus+Infection.">A Four-Antigen Mixture for Rapid Assessment of Onchocerca volvulus Infection.</a> PLoS Negl Trop Dis. 3, e438-e438 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+robust+luminescent+profiling+of+anti-La+and+other+autoantibodies+in+Sj+gren&#x2019;s+syndrome.">Sensitive and robust luminescent profiling of anti-La and other autoantibodies in Sj gren&#x2019;s syndrome.</a> Autoimmunity. 139, 241-245 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+antibody+quantification+and+generation+of+whole+proteome+antibody+response+profiles+using+LIPS+(luciferase+immunoprecipitation+systems).">Rapid antibody quantification and generation of whole proteome antibody response profiles using LIPS (luciferase immunoprecipitation systems).</a> Biochem Biophys Res Commun. 352, 889-895 (2007).</li><li>Burbelo, P. D., Groot, S., Dalakas, M. C., Iadarola, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+definition+profiling+of+autoantibodies+to+glutamic+acid+decarboxylases+GAD65/GAD67+in+stiff-person+syndrome.">High definition profiling of autoantibodies to glutamic acid decarboxylases GAD65/GAD67 in stiff-person syndrome.</a> Biochem Biophys Res Commun. 366, 1-7 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+luminescence+assay+for+autoantibodies+to+mammalian+cell-prepared+insulinoma-associated+protein+2.">A new luminescence assay for autoantibodies to mammalian cell-prepared insulinoma-associated protein 2.</a> Diabetes Care. 31, 1824-1826 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+diagnosis+of+human+herpes+simplex+virus+type+1+and+2+infections+by+luciferase+immunoprecipitation+system+assay.">Serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system assay.</a> Clin Vaccine Immunol. 16, 366-371 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+quantitative+serological+detection+of+anti-cytomegalovirus+(CMV)+antibodies.">Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies.</a> Virol J. 6, 45-45 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-antigen+mixture+containing+v-cyclin+for+serological+screening+of+human+herpesvirus+8+infection.">Four-antigen mixture containing v-cyclin for serological screening of human herpesvirus 8 infection.</a> Clin Vaccine Immunol. 16, 621-627 (2009).</li><li>Ramanathan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+luciferase+immunoprecipitation+systems+assay+enhances+the+sensitivity+and+specificity+of+diagnosis+of+Strongyloides+stercoralis+infection.">A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection.</a> J Infect Dis. 198, 444-451 (2008).</li></ol>

LABIOS requiere la optimización del ensayo mínimo y, debido a su simplicidad, los datos de alta calidad por lo general se pueden generar con los labios en menos de dos semanas de cualquier antígeno dado. Los pasos que más tiempo se encuentra la clonación y la generación del vector de expresión apropiado plásmido que contenía la fusión Ruc-antígeno. Una vez que estos plásmidos se generan, los antígenos individuales o múltiples puede ser rápidamente probado con los labios como se describió anteriormente. Cabe señalar que los labios es muy versátiles, que el suero puede ser probado en un formato de tubo, o bien en una placa de microtitulación de filtro con la ayuda de una estación de trabajo robotizada Biomek 2 o un lavador de placas de filtración al vacío. Este sistema altamente escalable permite a los labios paralelos perfil de un grupo de humanos autoantígenos 3 o el proteoma completo de un 4 virus. Es probable que muchos perfiles diferentes de anticuerpos generados por los labios serán de utilidad para el diagnóstico de 3-9, antígeno de descubrimiento 9, siguiendo el curso de un tratamiento de 10, el control de vacunas y tiene amplias implicaciones para los estados específicos de la enfermedad y la salud pública en general.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>HiSpeed Plasmid Midi KIt</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
<tr>
<td>100 x 20 mm tissue culture dishes</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>353003</td>
<td>&nbsp;</td>
<tr>
<td>Opti-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>FuGENE 6</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1814443 </td>
<td>&nbsp;</td>
<tr>
<td>Complete, Mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836153001</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 96 DeepWell 1 ml plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120 </td>
<td>Deep well plate with lid and seal with Parafilm</td>
</tr>
<tr>
<td>Polypropylene microtiter plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120</td>
<td>&nbsp;</td>
<tr>
<td>HTS Filter plate</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSBVN1B50</td>
<td>&nbsp;</td>
<tr>
<td>Ultralink Immobilized Protein A/G</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>53133 (10ml)</td>
<td>&nbsp;</td>
<tr>
<td>Renilla Luciferase Assay System</td>
<td>&nbsp;</td>
<td>Promega </td>
<td>E2820</td>
<td>For 2000 assays</td>
</tr>
<tr>
<td>Centro microplate luminometer</td>
<td>&nbsp;</td>
<td>Berthold</td>
<td>LB 960</td>
<td>&nbsp;</td>		</tbody>
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antibody profiling by luciferase immunoprecipitation systems (lips)

Tecnologías para la exhaustiva comprensión y cuantificación de perfiles de anticuerpos frente a autoantígenos y los agentes infecciosos pueden producir nuevos conocimientos sobre los mecanismos de la enfermedad y puede dilucidar los nuevos marcadores de la enfermedad substratify con diferentes características clínicas y comprender mejor la patogénesis. Hemos desarrollado un método altamente cuantitativa llamada luciferasa Sistemas de inmunoprecipitación (LIPS) para crear perfiles de los pacientes la respuesta de anticuerpos suero de autoantígenos y antígenos de patógenos asociados con la infección. A diferencia de las pruebas ELISA, los labios muy sensibles es fácil de implementar para estudiar los perfiles de respuesta serológica humoral a los antígenos diferentes en un formato universal y produce títulos de anticuerpos dinámica un alcance de hasta 6 log<sub&gt; 10</sub&gt; Para algunos antígenos. En estos estudios, el perfil cuantitativo por los labios del paciente la respuesta humoral contra los paneles de antígenos o incluso todo el proteoma de algunos patógenos (por ejemplo el VIH), suele ser más informativos que las pruebas de un solo antígeno por ELISA. Además, los labios también elimina el tiempo y el esfuerzo necesarios para producir antígenos altamente purificados, así como los pasos del ensayo de mano de obra necesaria para la optimización de ELISA estándar. Aquí les ofrecemos un protocolo detallado que describe los aspectos técnicos de la realización de ensayos de labios fácilmente perfiles de las respuestas de anticuerpos a los antígenos únicos o múltiples.

Watch this Scientific Journal Video about Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS at JoVE.com

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

Los aspectos técnicos de la realización de los labios (Sistemas de luciferasa inmunoprecipitación) se describen. El enfoque general consiste en la expresión de genes quiméricos que codifican antígenos fusionados a Renilla luciferase (RUC) en células de mamíferos. Crudo Ruc-antígeno extractos son preparados y, sin purificación, empleados en ensayos de inmunoprecipitación para cuantificar anticuerpos.

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 ...

antibody-profiling-by-luciferase-immunoprecipitation-systems-lips

Research

JoVE Journal

Biology

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

26.9K vistas.  National Institute of Dental and Craniofacial Research, National Institutes of Health. Los aspectos técnicos de la realización de los labios (Sistemas de luciferasa inmunoprecipitación) se describen. El enfoque general consiste en la expresión de genes quiméricos que codifican antígenos fusionados a Renilla luciferase (RUC) en células de mamíferos. Crudo Ruc-antígeno extractos son preparados y, sin purificación, empleados en ensayos de inmunoprecipitación para cuantificar anticuerpos.

Video: Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

AC electrocinéticas fenómenos generados por las Estructuras de microelectrodos

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

Investigación

Biología

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Inmunoprecipitación de ADN metilado

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP-FCM: inmunoprecipitación detectada por citometría de flujo

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

En la transcripción in vitro y la nivelación de mRNA de luciferasa Gaussia Seguido por transfección de células HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Anticuerpos, químicamente bloqueado Microarray de multiplexado de alto rendimiento de perfiles de glicosilación de proteínas específicas en muestras complejas

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Análisis de la Traducción Iniciación Durante condiciones de estrés por polisoma Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Perfiles de Células Madre Embrionarias Humanas individual por RT-PCR cuantitativa

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Evaluación del selectivo ARNm traducción en células de mamífero por polisoma perfiles

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

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

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

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

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