Ce travail a été financé par 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>

Ce travail a été fait à Luminex Corporation avec du matériel fabriqué à Luminex Corporation. R &amp; D Systems &amp; EMD Millipore sont des partenaires stratégiques de Luminex Corporation; licence pour développer et commercialiser des tests multiplex xMAP base.

La conversion d&#39;un test ELISA pour la plate-forme Luminex xMAP peuvent être aussi simples que la substitution streptavidine peroxydase de raifort (SA-HRP) dans un cadre typique kit ELISA avec de la streptavidine phycoérythrine (SA-PE), et l&#39;optimisation de la performance. Pour ceux qui veulent créer un immunoessai xMAP de la terre vers le haut, ce qui peut être accompli avec un protocole simple qui permet également l&#39;évaluation multiplex rapide, des paires d&#39;anticorps. Les réactifs pour le dosage xMAP ont été facilement préparés en utilisant le kit Anticorps xMAP couplage pour coupler les anticorps de capture désignés pour MagPlex faible concentration des microsphères. L&#39;utilisation d&#39;une faible concentration microsphères réduit le coût de l&#39;élaboration de tests, tout en offrant la performance de dosage même de concentration plus élevée des microsphères. Le montant de temps requis pour préparer couplé MagPlex microsphères est d&#39;environ 3 heures, ce qui est beaucoup plus rapide que les 22 - 24 heures nécessaires pour recouvrir le puits d&#39;une plaque ELISA, suivie d&#39;un traitementdes puits revêtus. La performance du test xMAP est également supérieure à la technique ELISA en termes de limite de détection (&lt;4 pg / ml vs&gt; 31 pg / mL) et dynamique large (&lt;4 pg / mL à&gt; 8000 pg / ml contre 16 pg / ml à 1000 pg / mL). Lecteurs de plaques ont une gamme limitée DO qui est soit 3 ou 4 DO, limitant la limite supérieure de la plage dynamique pour un dosage. Sans doute, tous les anticorps travailler dans un format ELISA et tous les anticorps qui fonctionnent bien dans un test ELISA sont facilement transférables au format de dosage xMAP. Toutefois, étant donné que des dosages xMAP peuvent être multiplexés (c&#39;est à dire, fonctionner simultanément), il est possible d&#39;évaluer la capture de plusieurs combinaisons et détection des anticorps en même temps d&#39;identifier la meilleure paire à utiliser pour un essai. Ce processus permet d&#39;économiser beaucoup de temps et réactifs par rapport à la procédure d&#39;élaboration ELISA, qui se limite à l&#39;évaluation d&#39;une paire à la fois. Si deux ou plusieurs paires d&#39;anticorps effectuer équivalente; d&#39;autres paramètres de l&#39;essai peutêtre considéré pour déterminer l&#39;aptitude de la paire (par exemple, disponibilité, coût, etc.) En plus de la performance du test et une flexibilité améliorées avec le dosage xMAP, il ya aussi d&#39;importantes économies. La quantité recommandée d&#39;anticorps nécessaire pour enrober un seul puits d&#39;une plaque ELISA est 400ng, alors que la quantité requise pour les billes utilisées dans un puits d&#39;un dosage xMAP est d&#39;environ 7,5 ng. Ainsi, la quantité d&#39;anticorps nécessaire pour un ELISA ainsi fournira plus de 50 résultats des tests, si elle est utilisée dans un test xMAP. Pour les applications impliquant des échantillons précieux, xMAP a également un avantage significatif. Le volume de l&#39;échantillon recommandé pour le test ELISA est de 100 pi tandis que le volume requis pour le dosage xMAP peuvent être la moitié de ce que, ou moins. En résumé, la conversion d&#39;un test ELISA pour la plate-forme Luminex xMAP est simple, efficace et économique, tout en produisant un essai avec une gamme dynamique supérieure et de la sensibilité.

<table border="1"><tbody><tr><td> Description </td><td> Vendeur </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Human TNF-α/TNFSF1A DuoSet kit ELISA </td><td> R &amp; D Systems </td><td> DY210 </td><td> Comprend monoclonaux et de la biotine couplés des anticorps polyclonaux et recombinant standards protéine TNF-α </td></tr><tr><td> Anticorps monoclonal anti-TNF-α </td><td> Abcam </td><td> Ab18696 </td><td> Anticorps de capture, clone CH8820 </td></tr><tr><td> Anticorps monoclonal anti-TNF-α </td><td> Abcam </td><td> Ab16166 </td><td> Anticorps Biotine détection couplé, clone AS1 </td></tr><tr><td> TNF alpha protéines </td><td> Abcam </td><td> Ab9642 </td><td> Recombinant norme protéine TNF-α </td></tr><tr><td> Anticorps monoclonal anti-TNF-α </td><td> Novus </td><td> NBP1-50115 </td><td> Anticorps de capture, clone 4H31 </td></tr><tr><td> Anticorps monoclonal anti-TNF-α </td><td> Novus </td><td> NB100-78162 </td><td> Anticorps Biotine détection couplé, clone MAb11 </td></tr><tr><td> TNF alpha protéines </td><td> Novus </td><td> NBC1-18460 </td><td> Recombinant norme protéine TNF-α </td></tr><tr><td> Anticorps monoclonal anti-TNF-α </td><td> EMD Millipore </td><td> MAB1141 </td><td> Anticorps de capture, cloner 3C7.2 </td></tr><tr><td> Anticorps polyclonaux anti-TNF-α </td><td> EMD Millipore </td><td> 654250 </td><td> Anticorps polyclonal de lapin de détection </td></tr><tr><td> Streptavidine-phycoérythrine </td><td> Mousse </td><td> SAPE-001 </td><td> Réactif fluorescent pour reporter test Luminex xMAP </td></tr><tr><td> MAGPIX w / Software XPONENT </td><td> Luminex Corporation </td><td> MAGPIX-XPONENT </td><td> Instrument Luminex </td></tr><tr><td> xMAP Anticorps Coupling (ABC) Kit </td><td> Luminex Corporation </td><td> 40-50016 </td><td> Comprend réactif EDC, sulfo-NHS réactif, tampon d&#39;activation, tampon de lavage, des tubes de 1,5 mL de réaction, et les pipettes jetables </td></tr><tr><td> MagPlex microsphères, à faible concentration </td><td> Luminex Corporation </td><td> MC10012-ID, MC10013-ID, MC10014-ID, MC10015-ID </td><td> Perles à faible concentration (@ 2,5 x 10 6 billes / mL) </td></tr><tr><td> EZ-Link Sulfo-NHS-LC-Biotine </td><td> Thermo Fisher </td><td> PI-21335 </td><td> Kit de détection des anticorps pour Biotinylation non modifiée </td></tr><tr><td> Tecan Infini F200 Lecteur </td><td> Tecan </td><td></td><td> Lecteur de plaque ELISA </td></tr><tr><td> Phosphate Buffered Saline </td><td> Sigma-Aldrich </td><td> P-3688 </td><td> 1% du PBS-BSATampon de dosage, </td></tr><tr><td> Une pinte-Cleaner Compact ultrasons, 115 VAC </td><td> Cole-Parmer </td><td> WU-08849-00 </td><td> Produire une fréquence de fonctionnement effective de 55 kHz </td></tr><tr><td> Séparateur magnétique Tube </td><td> Luminex Corporation </td><td> CN-0288-01 </td><td> Pour la séparation seul tube de 1,5 ml magnétique dans les étapes de lavage de couplage </td></tr><tr><td> Séparateur plaque magnétique </td><td> Luminex Corporation </td><td> CN-0269-01 </td><td> Pour 96-ainsi la séparation plaque magnétique dans les étapes de lavage de dosage </td></tr></tbody></table>

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

Le dosage immuno-enzymatique (ELISA) a longtemps été le principal outil pour la détection d&#39;analytes d&#39;intérêt dans des échantillons biologiques pour la recherche à la fois sciences de la vie et les diagnostics cliniques. Cependant, le test ELISA a ses limites. Il est généralement réalisé dans une microplaque de 96 puits, et les puits sont recouverts d&#39;anticorps de capture, ce qui nécessite une quantité relativement importante de l&#39;échantillon pour capturer un antigène d&#39;intérêt. La grande surface des puits et la liaison hydrophobe de l&#39;anticorps de capture peut également conduire à fond non spécifique contraignante et plus. En outre, la plupart des tests ELISA compter sur l&#39;intermédiaire d&#39;enzyme amplification du signal afin d&#39;obtenir une sensibilité raisonnable. D&#39;amplification telle n&#39;est pas toujours linéaire et peut fausser les résultats ainsi. Au cours des 15 dernières années, une nouvelle technologie a vu le jour qui offre les avantages de la technique ELISA, mais aussi permet un débit plus élevé, une plus grande souplesse, volume de l&#39;échantillon réduit, et à moindre coût, avec un wo similairerkflow 1, 2. Luminex xMAP de la technologie est une plate-forme de réseau de microsphères (billes) permettant à la fois monoplex et essais multiplex qui peuvent être appliquées à la fois des protéines et acides nucléiques applications 3-5. Les billes ont l&#39;anticorps de capture immobilisé de façon covalente sur une surface plus petite, ce qui nécessite moins anticorps de capture et les volumes d&#39;échantillons plus petites, par rapport à ELISA, et la liaison non spécifique est réduite de manière significative. Volumes d&#39;échantillons plus petits sont importantes lorsque l&#39;on travaille avec des échantillons limitant tels que le liquide céphalo-rachidien, du liquide synovial, etc 6. Multiplexage le dosage réduit encore le volume d&#39;échantillon exigences, permettant plusieurs résultats à partir d&#39;un seul échantillon. Les récentes améliorations de Luminex comprennent: le nouveau système MAGPIX, une plus petite, moins coûteuse, plus facile à utiliser l&#39;analyseur; faible concentration magnétique MagPlex microsphères qui éliminent le besoin de coûteux plaques filtrantes et viennent dans une concentration de travail mieux adapté pour le développement de tests et defaible débit demandes, et l&#39;anticorps xMAP accouplement (ABC) du kit qui comprend un protocole, les réactifs et produits consommables nécessaires pour coupler billes à l&#39;anticorps de capture d&#39;intérêt. (Voir la section Matériaux pour une liste détaillée du contenu du kit.) Dans cette expérience, nous convertissons un test pré-optimisée ELISA pour la cytokine TNF-alpha à ​​la plate-forme xMAP et comparer la performance des deux méthodes 7-11. TNF-alpha est un biomarqueur utilisé dans la mesure des réponses inflammatoires chez les patients souffrant de troubles auto-immunes. Nous commençons par le couplage quatre anticorps de capture candidats à quatre ensembles de microsphères ou des régions différentes. Lorsque mélangés, ces quatre ensembles de permettre l&#39;analyse simultanée de tous les quatre candidats ayant quatre anticorps de détection distinctes afin de déterminer la meilleure paire d&#39;anticorps, les réactifs d&#39;épargne, de l&#39;échantillon et le temps. Deux dosages xMAP sont ensuite construits avec les deux paires d&#39;anticorps les plus optimales et leur performance estpar rapport à celle du dosage ELISA originale en ce qui concerne la puissance du signal, la gamme dynamique, et la sensibilité.

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 test ELISA peut être facilement converti en un dosage Luminex xMAP et, grâce aux bienfaits de multiplexage, plusieurs anticorps peuvent être projetés simultanément pour identifier une paire d&#39;anticorps optimal, ce qui entraîne une sensibilité accrue et une gamme dynamique, tout en réduisant les coûts du test.

Conversion d&#39;un test ELISA de capture à un test Luminex xMAP utilisant une méthode de dépistage d&#39;anticorps 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 vues.  Luminex Corporation. Un test ELISA peut être facilement converti en un dosage Luminex xMAP et, grâce aux bienfaits de multiplexage, plusieurs anticorps peuvent être projetés simultanément pour identifier une paire d'anticorps optimal, ce qui entraîne une sensibilité accrue et une gamme dynamique, tout en réduisant les coûts du test.

Vidéo: 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.

Microdissection laser des tissus de mammifères

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

Recherche

Biologie

Cell Capture Using a Microfluidic Device

Capturez cellule à l&#39;aide d&#39;un dispositif microfluidique

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.

Méthode de coloration Mont anticorps entier de 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 ...

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.

Dépistage ARNi pour identifier les phénotypes postembryonnaires dans 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 test de dépistage fluorescent pour identifier des modulateurs de canaux 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.

Un essai en phase liquide d&#39;affinité de capture utilisant des billes magnétiques pour étudier interaction protéine-protéine: Le poliovirus Exemple-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.

Criblage à haut débit fonctionnelle utilisant une maison double lueur de dosage de luciférase

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.

Une méthode de sélection aptamères Structure commutation Appliqué à un colorimétrique Or nanoparticules 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.

Identification de paires Kinase-substrat en utilisant criblage à haut débit


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-enzyme préalable au moyen d&#39;un système de criblage enzymatique génétique à haut débit

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