Diese Arbeit wurde von Luminex Corporation finanziert.

<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>

Diese Arbeit wurde am Luminex Corporation mit Ausrüstung bei Luminex Corporation hergestellt getan. R &amp; D Systems &amp; EMD Millipore sind strategische Partner von Luminex Corporation, lizenziert zur Entwicklung und Vermarktung xMAP Multiplex Assays.

Die Umwandlung eines ELISA-Assays auf Basis der Luminex xMAP-Plattform kann so einfach sein wie Substitution Streptavidin Meerrettichperoxidase (SA-HRP) in einer typischen ELISA-Kit mit Streptavidin Phycoerythrin (SA-PE), und die Optimierung für die Leistung. Für diejenigen, die eine xMAP Immunoassay von Grund auf neu erstellen möchten, können dies mit einem einfachen Protokoll, das ermöglicht auch die schnelle, Multiplex-Auswertung der Antikörper-Paaren durchgeführt werden. Die Reagenzien für die xMAP Assay wurden leicht unter Verwendung des xMAP Antikörper Kupplungssatz zum Koppeln der Fänger-Antikörper bezeichnet, um MagPlex niedriger Konzentration Mikrokügelchen. Die Verwendung einer niedrigen Konzentration Mikrokügelchen verringert die Kosten der Assay-Entwicklung bei gleicher Testleistung höherer Konzentration Mikrokügelchen. Die Zeit, die erforderlich ist, um Mikrosphären gekoppelt MagPlex vorzubereiten, ist ca. 3 Stunden, das ist viel schneller als die 22 bis 24 Stunden erforderlich, um das Fell gut einer ELISA-Platte, gefolgt von einer Behandlungder beschichteten Vertiefungen. Die Leistung der xMAP-Versuch ist auch besser als der ELISA in Bezug auf die Nachweisgrenze (&lt;4 pg / ml vs&gt; 31 pg / ml) und der dynamische Bereich (&lt;4 pg / ml bis&gt; 8000 pg / ml vs 16 pg / ml bis 1000 pg / ml). Tafel Software haben eine begrenzte OD Bereich, der entweder 3 oder 4, OD, beschränken die obere Grenze des dynamischen Bereichs für einen Assay. Zweifellos wird nicht alle Antikörper in einem ELISA-Format zu arbeiten und nicht alle Antikörper, die gut funktionieren in einem ELISA sind leicht übertragbar auf die xMAP-Assay-Format. Da jedoch xMAP Assays können gemultiplext (dh, gleichzeitig ausgeführt werden), ist es möglich, mehrere Abscheidung und Nachweis-Antikörper-Kombinationen gleichzeitig zu evaluieren, das beste Paar zu identifizieren, für einen Assay zu verwenden. Dieses Verfahren spart erheblich Zeit und Reagenzien im Vergleich zu dem ELISA-Verfahren Entwicklung, die auf der Auswertung eines Paares in einer Zeit ist begrenzt. Wenn zwei oder mehr Antikörper-Paare ausführen äquivalent; andere Parameter des Assays könnenals Eignung des Paares (z. B. Verfügbarkeit, Kosten, usw.) zu bestimmen. Neben der verbesserten Assay-Performance und Flexibilität mit der xMAP-Assay, gibt es auch erhebliche Kosteneinsparungen. Die empfohlene Menge des Antikörpers zur Beschichtung eines einzigen Vertiefung eine ELISA-Platte ist 400 ng erforderlich ist, während die Menge der Kügelchen in einer Vertiefung einer xMAP Assay verwendet liegt bei ca. 7,5 ng. Somit wird die Menge des Antikörpers für ein ELISA benötigt auch wird mehr als 50 Testergebnisse, wenn es in einer xMAP Assay verwendet. Für Anwendungen mit wertvollen Proben, xMAP hat auch einen erheblichen Vorteil. Das Volumen der Probe für den ELISA empfohlen ist 100 ul während das Volumen für die xMAP-Assay erforderlich, dass die Hälfte oder weniger sein können. Zusammenfassend ist die Umwandlung eines ELISA-Assays auf Basis der Luminex xMAP-Plattform unkompliziert, effizient und Kosten sparend, während die Herstellung eines Assays mit überlegener Dynamik und Sensibilität.

<table border="1"><tbody><tr><td> Beschreibung </td><td> Verkäufer </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Menschliche TNF-α/TNFSF1A DuoSet ELISA-Kit </td><td> R &amp; D Systems </td><td> DY210 </td><td> Enthält monoklonale und polyklonale Biotin-gekoppelten Antikörpern und rekombinanten TNF-α-Protein-Standard </td></tr><tr><td> Monoklonaler Antikörper gegen TNF-α </td><td> Abcam </td><td> Ab18696 </td><td> Fänger-Antikörper, Klon CH8820 </td></tr><tr><td> Monoklonaler Antikörper gegen TNF-α </td><td> Abcam </td><td> Ab16166 </td><td> Biotin-gekoppelten Detektions-Antikörper, Klon AS1 </td></tr><tr><td> TNF-alpha-Protein </td><td> Abcam </td><td> Ab9642 </td><td> Rekombinanten TNF-α-Protein-Standard </td></tr><tr><td> Monoklonaler Antikörper gegen TNF-α </td><td> NOVUS </td><td> NBP1-50115 </td><td> Fänger-Antikörper, Klon 4H31 </td></tr><tr><td> Monoklonaler Antikörper gegen TNF-α </td><td> Novus </td><td> NB100-78162 </td><td> Biotin-gekoppelten Detektions-Antikörper, Klon MAb11 </td></tr><tr><td> TNF-alpha-Protein </td><td> Novus </td><td> NBC1-18460 </td><td> Rekombinanten TNF-α-Protein-Standard </td></tr><tr><td> Monoklonaler Antikörper gegen TNF-α </td><td> EMD Millipore </td><td> MAB1141 </td><td> Fänger-Antikörper, klonen 3C7.2 </td></tr><tr><td> Polyklonalen Antikörper gegen TNF-α </td><td> EMD Millipore </td><td> 654250 </td><td> Kaninchen polyklonaler Antikörper-Erkennung </td></tr><tr><td> Streptavidin-Phycoerythrin </td><td> Moos </td><td> SAPE-001 </td><td> Fluoreszierenden Reporter-Reagenz für die Luminex xMAP-Test </td></tr><tr><td> MAGPIX w / Xponent Software </td><td> Luminex Corporation </td><td> MAGPIX-Xponent </td><td> Luminex-Gerät </td></tr><tr><td> xMAP Antikörper-Kupplung (AbC) Kit </td><td> Luminex Corporation </td><td> 40-50016 </td><td> Beinhaltet EDC-Reagenz, Sulfo-NHS-Reagenz, Aktivierung, Waschpuffer, 1,5 ml Reaktionsgefäße und Einwegpipetten </td></tr><tr><td> MagPlex Microspheres, geringe Konzentration </td><td> Luminex Corporation </td><td> MC10012-ID, MC10013-ID, MC10014-ID, MC10015-ID </td><td> Kügelchen mit niedriger Konzentration (bei ​​2,5 x 10 6 Kügelchen / ml) </td></tr><tr><td> EZ-Link Sulfo-NHS-LC-Biotin </td><td> Thermo Fisher </td><td> PI-21335 </td><td> Biotinylierungskit für unmodifizierte Detektionsantikörper </td></tr><tr><td> Tecan Infinite F200 Reader </td><td> Tecan </td><td></td><td> ELISA-Reader </td></tr><tr><td> Phosphate Buffered Saline </td><td> Sigma-Aldrich </td><td> P-3688 </td><td> 1% PBS-BSA, Assay-Puffer </td></tr><tr><td> One-Pint Kompakte Ultraschall-Reiniger, 115 VAC </td><td> Cole-Parmer </td><td> WU-08849-00 </td><td> Produzieren Sie eine effektive Betriebsfrequenz von 55 kHz </td></tr><tr><td> Magnetische Rohr Separator </td><td> Luminex Corporation </td><td> CN-0288 bis 01 </td><td> Für einzelne 1,5 ml Röhrchen magnetische Trennung in Kopplung Waschschritte </td></tr><tr><td> Magnetische Plattenphasentrenner </td><td> Luminex Corporation </td><td> CN-0269 bis 01 </td><td> Für 96-Well-Platte magnetischen Trennung in Assay-Waschschritte </td></tr></tbody></table>

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

Die Enzyme Linked Immuno Assay (ELISA) ist seit langem das wichtigste Werkzeug für den Nachweis von Analyten in biologischen Proben für die biowissenschaftliche Forschung und klinische Diagnostik. Jedoch hat ELISA Einschränkungen. Es wird typischerweise in einer 96-Well-Mikrotiterplatte durchgeführt, und die Vertiefungen werden mit Einfang-Antikörper beschichtet, die eine relativ große Menge an Probe, um ein Antigen von Interesse zu erfassen. Die große Oberfläche der Vertiefungen und die hydrophobe Bindung der Fänger-Antikörper können auch auf nicht-spezifische Bindung und erhöhten Hintergrund führen. Darüber hinaus verlassen sich die meisten ELISAs auf Enzym-vermittelte Verstärkung des Signals, um eine angemessene Empfindlichkeit zu erreichen. Eine solche Verstärkung ist nicht immer linear und kann somit die Ergebnisse verfälschen. In den vergangenen 15 Jahren hat eine neue Technologie bietet sich heraus, dass die Vorteile des ELISA, sondern ermöglicht auch einen höheren Durchsatz, höhere Flexibilität, geringere Probenvolumen und niedrigeren Kosten, mit einer ähnlichen WOrkflow 1, 2. Luminex xMAP-Technologie ist eine Mikrosphären (Perle) Array-Plattform ermöglicht sowohl Monoplex und Multiplex-Assays, die sowohl für Protein-und Nukleinsäure-Anwendungen 5.3 angewendet werden können. Die Perlen haben den Capture-Antikörper kovalent an eine kleinere Oberfläche immobilisiert, die weniger Capture-Antikörper und kleiner Probenvolumina, verglichen mit ELISA und nicht-spezifische Bindung wird deutlich reduziert. Kleinere Probenmengen sind wichtig bei der Arbeit mit Grenzmuster wie Cerebrospinalflüssigkeit, Synovialflüssigkeit, etc. 6. Multiplexen der Test weiter reduziert Probenvolumen Anforderungen, so dass mehrere Ergebnisse aus einer einzigen Probe. Die jüngsten Verbesserungen von Luminex enthalten: die neue MAGPIX System, ein kleiner, billiger, einfacher zu bedienende Analysator; niedriger Konzentration Magnetic MagPlex Mikrokugeln, die den Bedarf an teuren Filterplatten eliminieren und kommen in einer Konzentration arbeiten besser für Assay-Entwicklung geeignet undLow-Throughput-Anwendungen, und die xMAP-Antikörper-Kupplung (AbC) Kit, das ein Protokoll, Reagenzien und Verbrauchsmaterialien, die für Kupplung Perlen an den Fänger-Antikörper von Interesse enthält. (Siehe Abschnitt Materialien für eine detaillierte Liste der Inhalte der.) In diesem Experiment, setzen wir eine voroptimierte ELISA-Assay für TNF-alpha Zytokine an der xMAP-Plattform und die Performance der beiden Methoden 7-11. TNF-alpha als ein Biomarker bei der Messung von entzündlichen Reaktionen bei Patienten mit Autoimmunkrankheiten verwendet. Wir beginnen mit der Kupplung vier Kandidaten Fänger-Antikörper zu vier verschiedene Mikrosphären-Sets oder Regionen. Wenn sie miteinander vermischt, erlauben diese vier Sätze für die gleichzeitige Prüfung aller vier Kandidaten mit vier getrennten Nachweis von Antikörpern gegen die besten Antikörper Paar, spart Reagenzien, Probe und Zeit zu bestimmen. Zwei xMAP Assays werden dann mit den beiden optimale Antikörper-Paare ausgebildet und ihre Wirkung istim Vergleich zu der ursprünglichen ELISA-Test in Bezug auf Signalstärke, Dynamikbereich und Empfindlichkeit.

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

Ein ELISA kann leicht zu einem Luminex xMAP Assay umgewandelt werden und durch die Vorteile der Multiplexen mehrere Antikörper gleichzeitig gescreent werden, um eine optimale Antikörperpaar zu identifizieren, was zu einer erhöhten Empfindlichkeit und den Dynamikbereich, während reduzierenden Assay einsehbar.

Umbau eines Capture-ELISA zu einem Luminex xMAP Assay unter Verwendung eines Multiplex-Antikörper-Screening-Methode

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 Aufrufe.  Luminex Corporation. Ein ELISA kann leicht zu einem Luminex xMAP Assay umgewandelt werden und durch die Vorteile der Multiplexen mehrere Antikörper gleichzeitig gescreent werden, um eine optimale Antikörperpaar zu identifizieren, was zu einer erhöhten Empfindlichkeit und den Dynamikbereich, während reduzierenden Assay einsehbar.

Serie: 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 Capture Mikrodissektion von Säugetiergewebe

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

Forschung

Biologie

Cell Capture Using a Microfluidic Device

Zell-Capture mit einer mikrofluidischen Vorrichtung

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.

Verfahren zur Whole Mount Antikörper-Färbung in Küken

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-Screening auf postembryonalen Phänotypen in Identifizieren 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.

Ein fluoreszierendes Screening Assay zur Identifizierung von Modulatoren der GIRK Kanäle

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.

Eine flüssige Phase Affinity Capture Assay Verwendung von magnetischen Kügelchen, um Protein-Protein Interaktion Studie: Das Poliovirus-Nanobody Beispiel

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.

Hochdurchsatz-Screening mit einem Funktionshomemade Dual-Luciferase Assay-Schein

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.

Ein Verfahren zur Auswahl Struktur-Schalt Aptamere zu einem Farbmetrik Gold-Nanopartikeln Assay angewandt

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.

Identifikation von Kinase-Substrat-Pairs Mit 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).

Screening Multi-Enzym ein High-throughput Genetic Enzymscreening-System verwenden

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