Este trabalho foi financiado pela 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 trabalho foi feito em Luminex Corporation com equipamentos fabricados no Luminex Corporation. R &amp; D Systems &amp; EMD Millipore são parceiros estratégicos da Luminex Corporation; licenciada para desenvolver e comercializar ensaios multiplex xMAP base.

A conversão de um ensaio de ELISA para a plataforma xMAP Luminex pode ser tão simples como a substituição de estreptavidina peroxidase de rábano (SA-HRP) em um típico kit de ELISA com estreptavidina ficoeritrina (SA-PE), e optimizar o desempenho. Para aqueles que deseja criar um imunoensaio xMAP de baixo para cima, isto pode ser realizado com um protocolo simples, que também permite a avaliação multiplex rápido, um dos pares de anticorpos. Os reagentes para o ensaio xMAP foram facilmente preparado utilizando o kit de Anticorpo xMAP acoplamento para acoplar os anticorpos de captura designados para MagPlex baixa concentração microesferas. O uso de baixa concentração de microesferas reduz o custo do desenvolvimento do ensaio enquanto fornece o desempenho do ensaio mesmo de maior concentração microesferas. A quantidade de tempo necessário para preparar acoplado MagPlex microesferas é de aproximadamente 3 horas, o que é muito mais rápido do que o 22 - 24 horas necessário para revestir o poço de uma placa de ELISA, seguido de tratamentodos poços revestidos. O desempenho do ensaio xMAP é também superior ao ELISA em termos de limite de detecção (&lt;4 pg / mL vs&gt; 31 pg / mL) e gama dinâmica (&lt;4 pg / mL a&gt; 8000 pg / mL vs 16 pg / mL a 1000 pg / mL). Leitores de placas têm uma gama limitada OD que é 3 ou 4 OD, restringindo o limite superior da gama dinâmica para um ensaio. Sem dúvida, nem todos os anticorpos vai trabalhar em um formato de ELISA e nem todos os anticorpos que funcionam bem em um ELISA são facilmente transferíveis para o formato de ensaio xMAP. No entanto, uma vez que os ensaios xMAP podem ser multiplexados (isto é, executar simultaneamente), é possível avaliar a captura e várias combinações de anticorpos de detecção em simultâneo para identificar a melhor par a ser usado para um ensaio. Este processo poupa tempo significativo e reagentes em comparação com o processo de desenvolvimento de ELISA, que é limitado para a avaliação de um par de cada vez. No caso de dois ou mais pares de anticorpos realizar equivalentemente; outros parâmetros do ensaio podeser considerado para determinar a adequação do par (por exemplo, a disponibilidade, custo, etc.) Além disso para o desempenho do ensaio melhorado e flexibilidade com o ensaio xMAP, há também poupanças de custos significativas. A quantidade recomendada de anticorpo necessária para revestir um único poço de uma placa de ELISA é 400ng, enquanto que a quantidade requerida para as esferas utilizadas em um poço de um ensaio de xMAP é aproximadamente 7,5 ng. Assim, a quantidade de anticorpo necessária para um ELISA bem irá fornecer mais de 50 resultados do teste, se for utilizado num ensaio xMAP. Para aplicações que envolvam amostras preciosos, xMAP também tem uma vantagem significativa. O volume da amostra recomendado para o ELISA é de 100 L ao passo que o volume necessário para o ensaio xMAP pode ser metade do que, ou menos. Em resumo, a conversão de um ensaio de ELISA para a plataforma xMAP Luminex é simples, eficiente e de economia de custos, enquanto a produção de um ensaio com uma gama dinâmica superior e sensibilidade.

<table border="1"><tbody><tr><td> Descrição </td><td> Vendedor </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> Human TNF-α/TNFSF1A DuoSet ELISA kit </td><td> R &amp; D Systems </td><td> DY210 </td><td> Inclui monoclonais e biotina acoplados anticorpos policlonais e recombinante padrão de proteína TNF-α </td></tr><tr><td> Anticorpo monoclonal para o TNF-α </td><td> Abcam </td><td> Ab18696 </td><td> Captura de anticorpos, clone CH8820 </td></tr><tr><td> Anticorpo monoclonal para o TNF-α </td><td> Abcam </td><td> Ab16166 </td><td> Biotina anticorpo de detecção acoplado, o clone AS1 </td></tr><tr><td> TNF proteína alfa </td><td> Abcam </td><td> Ab9642 </td><td> Recombinante padrão de proteína TNF-α </td></tr><tr><td> Anticorpo monoclonal para o TNF-α </td><td> Novus </td><td> NBP1-50115 </td><td> Captura de anticorpos, clone 4H31 </td></tr><tr><td> Anticorpo monoclonal para o TNF-α </td><td> Novus </td><td> NB100-78162 </td><td> Biotina anticorpo de detecção acoplado, o clone MAb11 </td></tr><tr><td> TNF proteína alfa </td><td> Novus </td><td> NBC1-18460 </td><td> Recombinante padrão de proteína TNF-α </td></tr><tr><td> Anticorpo monoclonal para o TNF-α </td><td> EMD Millipore </td><td> MAB1141 </td><td> Captura de anticorpos, clonar 3C7.2 </td></tr><tr><td> Anticorpo policlonal para o TNF-α </td><td> EMD Millipore </td><td> 654250 </td><td> Coelho policlonal anticorpo de detecção </td></tr><tr><td> Estreptavidina-Ficoeritrina </td><td> Musgo </td><td> SAPE-001 </td><td> O reagente de repórter fluorescente para Luminex Assay xMAP </td></tr><tr><td> MAGPIX w / Software Xponent </td><td> Luminex Corporation </td><td> MAGPiX Xponent- </td><td> Instrumento Luminex </td></tr><tr><td> Anticorpo xMAP Coupling (ABC) Kit </td><td> Luminex Corporation </td><td> 40-50016 </td><td> Inclui EDC reagente, Sulfo-NHS reagente, tampão de activação, o tampão de lavagem, 1,5 mL tubos de reacção, e pipetas descartáveis </td></tr><tr><td> MagPlex Microesferas, baixa concentração </td><td> Luminex Corporation </td><td> MC10012-ID, MC10013-ID, MC10014-ID, MC10015-ID </td><td> Baixos grânulos de concentração (@ 2,5 x 10 6 grânulo / mL) </td></tr><tr><td> EZ-Link Sulfo-NHS-LC-biotina </td><td> Thermo Fisher </td><td> PI-21.335 </td><td> Kit Biotinilação para detecção de anticorpos não modificada </td></tr><tr><td> Tecan Infinito F200 Leitor </td><td> Tecan </td><td></td><td> Leitor de placas ELISA </td></tr><tr><td> Tampão fosfato salino </td><td> Sigma-Aldrich </td><td> P-3688 </td><td> 1% de PBS-BSA, Tampão de Ensaio </td></tr><tr><td> Uma Pint-Cleaner Compact Ultrasonic, 115 VAC </td><td> Cole-Parmer </td><td> WU-08.849-00 </td><td> Produzir uma freqüência de operação efetiva de 55 kHz </td></tr><tr><td> Separador Tubo Magnético </td><td> Luminex Corporation </td><td> CN-0288-01 </td><td> Para a separação único tubo 1,5 ml magnética em passos de lavagem de acoplamento </td></tr><tr><td> Separador de placa magnética </td><td> Luminex Corporation </td><td> CN-0269-01 </td><td> Para a separação de 96 poços da placa magnética em passos de lavagem de ensaio </td></tr></tbody></table>

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

O ensaio imunoenzimático (ELISA) tem sido a principal ferramenta para a detecção de analitos de interesse em amostras biológicas, tanto para pesquisas de ciências humanas e diagnósticos clínicos. Contudo, Elisa tem limitações. É tipicamente realizada em uma microplaca de 96 poços, e os poços são revestidos com anticorpo de captura, exigindo uma quantidade relativamente grande de amostra para capturar um antigénio de interesse. A grande área de superfície dos poços ea ligação hidrofóbica de anticorpo de captura pode também conduzir a não-específica ligação de fundo e aumentada. Além disso, a maioria dos ELISAs dependem mediada por enzima de amplificação de sinal, a fim de alcançar a sensibilidade razoável. Tal amplificação não é sempre linear e pode distorcer resultados assim. Nos últimos 15 anos, uma nova tecnologia surgiu, que oferece os benefícios do ELISA, mas também permite um maior rendimento, maior flexibilidade, volume da amostra reduzida, e menor custo, com um wo semelhanterkflow 1, 2. Luminex xMAP A tecnologia é uma plataforma de micro-array (pérola), permitindo tanto monoplex e ensaios multiplex que podem ser aplicadas tanto para proteína e aplicações de ácido nucléico 3-5. Os grânulos têm o anticorpo de captura covalentemente imobilizada sobre uma área de superfície menor, exigindo menos anticorpo de captura e um menor volume de amostra, em comparação com ELISA, e ligação não específica é significativamente reduzida. Volumes menores de amostra são importantes quando se trabalha com amostras limitantes, como líquor, líquido sinovial, etc 6. Multiplexação o ensaio reduz ainda mais os requisitos de volume de amostra, permitindo vários resultados de uma única amostra. Recentes melhorias por Luminex incluem: o sistema de MAGPIX novo, uma menor, menos caro, analisador mais fácil de usar; de baixa concentração Magnética MagPlex Microesferas que eliminam a necessidade de dispendiosos placas de filtro e vêm em uma concentração de trabalho mais adequado para o desenvolvimento de ensaios ede baixo rendimento aplicações; eo anticorpo xMAP Acoplamento Kit (ABC), que inclui um protocolo, os reagentes e produtos de consumo necessários para o acoplamento pérolas para o anticorpo de captura de interesse. (Veja a seção Materiais para uma lista detalhada do conteúdo do kit.) Nesta experiência, nós converter um pré-optimizado ensaio ELISA para TNF-alfa citocina para a plataforma xMAP e comparar o desempenho dos dois métodos 7-11. TNF-alfa é um biomarcador utilizado na medição das respostas inflamatórias em pacientes com doenças auto-imunes. Começamos por acoplamento quatro anticorpos de captura candidatos para quatro conjuntos de microesferas ou regiões diferentes. Quando misturado em conjunto, estes quatro conjuntos permitir o teste simultâneo de todos os quatro candidatos com quatro anticorpos de detecção separado para determinar a melhor par de anticorpos, os reagentes de poupança, amostra e do tempo. Dois ensaios xMAP são então construído com os dois pares de anticorpos mais óptimas e seu desempenho écomparada com a do ensaio de ELISA original em relação à intensidade do sinal, a gama dinâmica, e sensibilidade.

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

Um ELISA pode ser facilmente convertido para um ensaio de xMAP Luminex e, através dos benefícios da multiplexação, vários anticorpos podem ser rastreados simultaneamente para identificar um par de anticorpos óptima, resultando em aumento da sensibilidade e gama dinâmica, enquanto reduz o custo do ensaio.

A conversão de um ELISA de captura para um ensaio de xMAP Luminex usando um método de rastreio de anticorpos Multiplex

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 visualizações.  Luminex Corporation. Um ELISA pode ser facilmente convertido para um ensaio de xMAP Luminex e, através dos benefícios da multiplexação, vários anticorpos podem ser rastreados simultaneamente para identificar um par de anticorpos óptima, resultando em aumento da sensibilidade e gama dinâmica, enquanto reduz o custo do ensaio.

Vídeo: 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.

Laser Microdissecção Captura de tecidos 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 ...

Biologia

Cell Capture Using a Microfluidic Device

Captura de célula usando um dispositivo micro

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 coloração Whole Mount anticorpos em Pintainho

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 triagem para identificar fenótipos pós-embrionário em 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.

Um ensaio de rastreio fluorescente para identificação Moduladores de Canais 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.

A fase líquida ensaio de captura de afinidade usando pérolas magnéticas para estudar interação proteína-proteína: O Exemplo Poliovírus Nanobody-

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.

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

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

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.

Um método para selecionar Aptamers de comutação Estrutura Aplicado a um colorimétrico nanopartículas de ouro Assay

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.

Identificação de pares Kinase-substrato utilizando High Throughput Screening


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

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

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

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

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

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