Este trabajo fue financiado por Luminex Corporation.

<ol>
	<li>Fulton, J. R., McDade, R. L., Smith, P. L., Kienker, L. J., Kettman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+multiplexed+analysis+with+the+FlowMetrix+system.">Advanced multiplexed analysis with the FlowMetrix system.</a> Clinical Chemistry. 43, 1749-1756 (1997).</li><li>Carson, R. T., Vignali, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantation+of+15+cytokines+using+a+multiplexed+flow+cytometric+assay.">Simultaneous quantation of 15 cytokines using a multiplexed flow cytometric assay.</a> J. Immunol. Methods. 227, 41-52 (1999).</li><li>Peck, D., Crawford, E. D., Ross, K. N., Stegmaier, K., Golub, T. R., Lamb, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high-throughput+gene+expression+signature+analysis.">A method for high-throughput gene expression signature analysis.</a> Genome Biol. 7, 61 (2006).</li><li>VanDerMeid, K. R., Su, S. P., Krenzer, K. L., Ward, K. W., Zhang, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+extract+cytokines+and+matrix+metalloproteinases+from+Schirmer+strips+and+analyze+using+Luminex.">A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex.</a> Mol Vis. 17, 1056-1063 (2011).</li><li>Liu, J., Kibiki, G., Maro, V., Maro, A., Kumburu, H., Swai, N., Taniuchi, M., Gratz, J., Toney, D., Kang, G., Houpt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+reverse+transcription+PCR+Luminex+assay+for+detection+and+quantitation+of+viral+agents+of+gastroenteritis.">Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis.</a> J. Clin. Virol. 50, 308-313 (2011).</li><li>de Jager, W., Prakken, B. J., Bijlsma, J., WJ Kuis, W., Rijkers, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+multiplex+immunoassay+performance+in+human+plasma+and+synovial+fluid+following+removal+of+interfering+heterophilic+antibodies.">Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies.</a> J. Immunol. Methods. 300, 124-135 (2005).</li><li>Codorean, E., Nichita, C., Albulescu, L., Raducan, E., Popescu, I. D., Lonita, A. C., Albulescu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+xMAP+and+ELISA+cytokine+profiles;+development+and+validation+for+immunotoxicological+studies+in+vitro.">Correlation of xMAP and ELISA cytokine profiles; development and validation for immunotoxicological studies in vitro.</a> Roum. Arch. Microbiol. Immunol. 69, 3-19 (2010).</li><li>de Jager, W., te Velthuis, H., Prakken, B. J., Kuis, W., Rijkers, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+15+human+cytokines+in+a+single+sample+of+stimulated+peripheral+blood+mononuclear+cells.">Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells.</a> Clin. Diagn. Lab. Immunol. 10, 133-139 (2003).</li><li>DuPont, N. C., Wang, K. H., Wadhwa, P. D., Culhane, J. F., Nelson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+comparison+of+Luminex+multiplex+cytokine+analysis+kits+with+ELISA:+Determinations+of+a+panel+of+nine+cytokines+in+clinical+sample+culture+supernatants.">Validation and comparison of Luminex multiplex cytokine analysis kits with ELISA: Determinations of a panel of nine cytokines in clinical sample culture supernatants.</a> J. Reprod. Immunol. 66, 175-191 (2005).</li><li>Richens, J. L., Urbanowicz, R. A., Metcalf, R., Corne, J., O'Shea, P., Fairclough, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+validation+and+comparison+of+multiplex+cytokine+kits.">Quantitative validation and comparison of multiplex cytokine kits.</a> Journal of Biomolecular Screening. 15, 562-568 (2010).</li><li>Rizzi, G., Zhang, Y. J., Latek, R., Weiner, R., Rhyne, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+development+of+a+Luminex-based+assay+for+the+detection+of+human+IL-23.">Characterization and development of a Luminex-based assay for the detection of human IL-23.</a> Bioanalysis. 2, 1561-1572 (2010).</li></ol>

Este trabajo fue realizado a Luminex Corporation con equipos fabricados en el Luminex Corporation. R &amp; D Systems y EMD Millipore son socios estratégicos de Luminex Corporation, con licencia para desarrollar y comercializar los ensayos multiplex xMAP base.

La conversión de un ensayo ELISA para la plataforma Luminex xMAP puede ser tan simple como la sustitución de estreptavidina peroxidasa de rábano (SA-HRP) en un típico kit de ELISA con estreptavidina ficoeritrina (SA-PE), y la optimización del rendimiento. Para aquellos que desean crear un inmunoensayo xMAP desde cero, esto se puede lograr con un protocolo sencillo que también permite la evaluación rápida, múltiplex de los pares de anticuerpos. Los reactivos para el ensayo xMAP se prepararon fácilmente con el kit de anticuerpos xMAP de acoplamiento para acoplar los anticuerpos de captura designados para MagPlex baja concentración de microesferas. El uso de microesferas de baja concentración se reduce el coste de desarrollo de ensayos mientras que proporciona el rendimiento del ensayo mismo de mayor concentración de microesferas. La cantidad de tiempo requerido para preparar acoplado MagPlex microesferas es aproximadamente 3 horas, que es mucho más rápido que el 22 - 24 horas requerido para recubrir el pocillo de una placa de ELISA, seguido por el tratamientode los pocillos recubiertos. El rendimiento del ensayo xMAP es también superior a la de ELISA en términos de límite de detección (&lt;4 pg / ml vs&gt; 31 pg / ml) y rango dinámico (&lt;4 pg / ml a&gt; 8000 pg / ml frente a 16 pg / ml a 1000 pg / ml). Lectores de la placa tienen una limitada gama de DO que es 3 o 4 OD, restringiendo el límite superior del rango dinámico para un ensayo. Sin duda, no todos los anticuerpos funcionan en un formato ELISA y no todos los anticuerpos que funcionan bien en un método de ELISA son fácilmente transferibles a el formato de ensayo xMAP. Sin embargo, puesto que los ensayos de xMAP pueden ser multiplexados (es decir, ejecutar simultáneamente), es posible evaluar la captura de varias combinaciones de detección de anticuerpos y simultáneamente para identificar el mejor par de usar para un ensayo. Este proceso ahorra tiempo y reactivos en comparación con el procedimiento de desarrollo de ELISA, que está limitada a la evaluación de un par a la vez. Si dos o más pares de anticuerpos realizar equivalentemente; otros parámetros del ensayo puedeconsiderarse para determinar la idoneidad del par (por ejemplo, disponibilidad, coste, etc.) Además de un rendimiento mejorado y ensayo de flexibilidad con el ensayo xMAP, también hay un ahorro significativo. La cantidad recomendada de anticuerpo necesaria para recubrir un solo pozo de una placa de ELISA es 400ng, mientras que la cantidad necesaria para las perlas utilizadas en un pocillo de un ensayo xMAP es de aproximadamente 7,5 ng. Así, la cantidad de anticuerpo necesaria para un ELISA bien proporcionará más de 50 resultados de la prueba, si se utiliza en un ensayo xMAP. Para aplicaciones que implican muestras preciosas, xMAP también tiene una ventaja significativa. El volumen de muestra recomendado para el ELISA es 100 l, mientras que el volumen requerido para el ensayo xMAP puede ser un medio de que, o menos. En resumen, la conversión de un ensayo de ELISA para la plataforma Luminex xMAP es sencillo, eficiente y ahorro de costes, mientras que la producción de un ensayo con un rango dinámico superior y la sensibilidad.

<table border="1"><tbody><tr><td> Descripción </td><td> Vendedor </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Humanos TNF-α/TNFSF1A DuoSet kit ELISA </td><td> R &amp; D Systems </td><td> DY210 </td><td> Incluye monoclonales y biotina acoplados anticuerpos policlonales y proteína recombinante estándar de TNF-α </td></tr><tr><td> Anticuerpo monoclonal a TNF-α </td><td> Abcam </td><td> Ab18696 </td><td> Captura de anticuerpos, el clon CH8820 </td></tr><tr><td> Anticuerpo monoclonal a TNF-α </td><td> Abcam </td><td> Ab16166 </td><td> Biotina acoplado Detección de anticuerpos, el clon AS1 </td></tr><tr><td> La proteína TNF alfa </td><td> Abcam </td><td> Ab9642 </td><td> TNF-α recombinante de proteínas estándar </td></tr><tr><td> Anticuerpo monoclonal a TNF-α </td><td> Novus </td><td> NBP1-50115 </td><td> Captura de anticuerpos, el clon 4H31 </td></tr><tr><td> Anticuerpo monoclonal a TNF-α </td><td> Novus </td><td> NB100-78162 </td><td> Biotina acoplado Detección de anticuerpos, el clon MAb11 </td></tr><tr><td> La proteína TNF alfa </td><td> Novus </td><td> NBC1-18460 </td><td> TNF-α recombinante de proteínas estándar </td></tr><tr><td> Anticuerpo monoclonal a TNF-α </td><td> EMD Millipore </td><td> MAB1141 </td><td> Captura de anticuerpos, clonar 3C7.2 </td></tr><tr><td> Anticuerpos policlonales a TNF-α </td><td> EMD Millipore </td><td> 654250 </td><td> Detección de anticuerpo de conejo policlonal </td></tr><tr><td> Estreptavidina-ficoeritrina </td><td> Musgo </td><td> SAPE-001 </td><td> Reactivo fluorescente reportero de Luminex xMAP Ensayo </td></tr><tr><td> MAGPIX w / Software XPONENT </td><td> Luminex Corporation </td><td> MAGPIX-XPONENT </td><td> Aparato Luminex </td></tr><tr><td> xMAP anticuerpos de acoplamiento (ABC) Kit </td><td> Luminex Corporation </td><td> 40-50016 </td><td> Incluye reactivo de la EDC, el reactivo sulfo-NHS, el tampón de activación, el tampón de lavado, tubos de 1,5 mL de reacción y pipetas desechables </td></tr><tr><td> MagPlex microesferas, la baja concentración </td><td> Luminex Corporation </td><td> MC10012-ID, ID-MC10013, MC10014-ID, MC10015-ID </td><td> Cuentas de baja concentración (2,5 x @ 10 6 bolas / ml) </td></tr><tr><td> EZ-Link sulfo-NHS-LC-biotina </td><td> Thermo Fisher </td><td> PI-21335 </td><td> Biotinilación kit para la detección de anticuerpos sin modificar </td></tr><tr><td> Tecan Infinite F200 Reader </td><td> Tecan </td><td></td><td> Lector de placas ELISA </td></tr><tr><td> Tampón fosfato salino </td><td> Sigma-Aldrich </td><td> P-3688 </td><td> 1% de PBS-BSA, Tampón de ensayo </td></tr><tr><td> Una pinta-limpiador ultrasónico compacto, 115 VCA </td><td> Cole-Parmer </td><td> WU-08.849-00 </td><td> Produce una frecuencia de operación efectiva de 55 kHz </td></tr><tr><td> Separador de tubo magnético </td><td> Luminex Corporation </td><td> CN-0288 a 01 </td><td> Para la separación solo tubo 1,5 ml magnético en los pasos de lavado de acoplamiento </td></tr><tr><td> Separador placa magnética </td><td> Luminex Corporation </td><td> CN-0269-01 </td><td> Para 96-así separación placa magnética en los pasos de lavado de ensayo </td></tr></tbody></table>

conversion of a capture elisa to a luminex xmap assay using a multiplex antibody screening method

La prueba de inmunoensayo ligado a enzimas (ELISA) ha sido durante mucho tiempo la principal herramienta para la detección de analitos de interés en las muestras biológicas tanto para la investigación en ciencias biológicas y diagnóstico clínico. Sin embargo, ELISA tiene sus limitaciones. Se realiza típicamente en una microplaca de 96 pocillos, y los pocillos se revisten con anticuerpo de captura, que requiere una cantidad relativamente grande de la muestra para capturar un antígeno de interés. La gran superficie de los pocillos y la unión hidrofóbica de anticuerpo de captura puede también conducir a no específica la unión de fondo y mayor. Además, la mayoría de ELISA depender enzima mediada por la amplificación de la señal con el fin de lograr una sensibilidad razonable. Tal amplificación no siempre es lineal y puede por lo tanto los resultados de inclinación. En los últimos 15 años, una nueva tecnología ha surgido, que ofrece los beneficios de la prueba ELISA, sino que también permite un mayor rendimiento, mayor flexibilidad, reducción del volumen de la muestra, y un menor coste, con un ¡ay similaresrkflow 1, 2. Luminex xMAP Technology es una microesfera (bola) plataforma matriz que permite tanto monoplex y ensayos multiplex que se pueden aplicar tanto a la proteína y las aplicaciones de ácido nucleico 3-5. Las perlas tienen el anticuerpo de captura covalentemente inmovilizado sobre una superficie más pequeña, requiriendo menos anticuerpo de captura y pequeños volúmenes de muestra, en comparación con el ELISA, y la unión no específica se reduce significativamente. Pequeños volúmenes de muestra son importantes cuando se trabaja con muestras limitantes como el líquido cefalorraquídeo, líquido sinovial, etc 6. Multiplexación el ensayo reduce aún más los requisitos de volumen de la muestra, lo que permite múltiples resultados de una sola muestra. Las recientes mejoras de Luminex incluyen: el nuevo sistema de MAGPIX, uno más pequeño, menos costoso, más fácil de usar analizador; baja concentración magnética MagPlex microesferas que eliminan la necesidad de costosas placas de filtro y vienen en una concentración de trabajo más adecuado para el desarrollo y ensayobajo rendimiento de aplicaciones, y el anticuerpo xMAP kit de acoplamiento (ABC), que incluye un protocolo, reactivos y consumibles necesarios para el acoplamiento de cuentas para el anticuerpo de captura de interés. (Vea la sección de Materiales para obtener una lista detallada del contenido del kit.) En este experimento, convertir un ensayo de pre-optimizado ELISA para TNF-alfa citoquina a la plataforma xMAP y comparar el rendimiento de los dos métodos 7-11. TNF-alfa es un marcador biológico utilizado en la medición de las respuestas inflamatorias en pacientes con trastornos autoinmunes. Comenzamos por el acoplamiento de cuatro anticuerpos de captura candidatos a cuatro conjuntos de microesferas y regiones diferentes. Cuando se mezclan, estos cuatro conjuntos de permitir el análisis simultáneo de todos los cuatro candidatos con cuatro anticuerpos de detección separadas para determinar el mejor par de anticuerpos, reactivos de ahorro, la muestra y el tiempo. Dos ensayos xMAP luego se construye con los dos pares de anticuerpos más óptimos y es su rendimientoen comparación con la del original ensayo ELISA en cuanto a intensidad de la señal, el rango dinámico, y la sensibilidad.

Watch this Scientific Journal Video about Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method at JoVE.com

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

Un ELISA se puede convertir fácilmente a un ensayo de Luminex xMAP y, a través de los beneficios de anticuerpos de multiplexación, varios pueden ser examinados simultáneamente para identificar un par de anticuerpos óptima, dando lugar a una mayor sensibilidad y rango dinámico, mientras que reduce el coste del ensayo.

La conversión de un ELISA de captura para un ensayo de Luminex xMAP utilizando un anticuerpo Multiplex Método de detección

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

conversion-capture-elisa-to-luminex-xmap-assay-using-multiplex

Research

JoVE Journal

Biology

43.4K vistas.  Luminex Corporation. Un ELISA se puede convertir fácilmente a un ensayo de Luminex xMAP y, a través de los beneficios de anticuerpos de multiplexación, varios pueden ser examinados simultáneamente para identificar un par de anticuerpos óptima, dando lugar a una mayor sensibilidad y rango dinámico, mientras que reduce el coste del ensayo.

Video: Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Láser microdisección de captura de tejidos de mamíferos

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Investigación

Biología

Cell Capture Using a Microfluidic Device

Captura de células utilizando un dispositivo de microfluidos

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para la tinción de anticuerpos enteros en el monte del polluelo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de detección para identificar fenotipos postembrionarios en C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

Un ensayo de cribado para la identificación de moduladores fluorescente de canales GIRK

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Una afinidad líquido fase de captura de ensayo utilizando partículas magnéticas para estudiar la interacción proteína-proteína: el ejemplo de poliomyelits-Nanobodies

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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

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

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

Selección de alto rendimiento funcional utilizando un doble resplandor de ensayo de luciferasa Homemade

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

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Un método para la selección de aptámeros Estructura de conmutación Aplicado a una de nanopartículas de oro ensayo colorimétrico

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

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

Identificación de los pares quinasa-sustrato Usando selección de alto rendimiento


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

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

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

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

Multienzimático de detección sistemática mediante un sistema de cribado genético de la enzima de alto rendimiento

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