Questo lavoro è stato finanziato da 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>

Questo lavoro è stato fatto a Luminex Corporation con attrezzature prodotti presso Luminex Corporation. R &amp; D Systems &amp; EMD Millipore sono partner strategici di Luminex Corporation, la licenza per sviluppare e commercializzare saggi xMAP basati multiplex.

La conversione di un saggio ELISA per la piattaforma Luminex xMAP può essere semplice come sostituzione streptavidina perossidasi di rafano (HRP-SA) in una tipica kit ELISA con streptavidina ficoeritrina (SA-PE), e per ottimizzare le prestazioni. Per coloro che vogliono creare un immunodosaggio xMAP dalla terra in su, questo può essere realizzato con un protocollo semplice che consente anche il digiuno, la valutazione multiplex delle coppie di anticorpi. I reagenti per il dosaggio xMAP erano facilmente preparati usando il kit anticorpo xMAP accoppiamento per accoppiare gli anticorpi di cattura designati a concentrazione microsfere MagPlex basso. L&#39;uso di microsfere concentrazione bassa riduce il costo di sviluppo test fornendo le stesse prestazioni di dosaggio microsfere concentrazione superiore. La quantità di tempo necessario per preparare accoppiato MagPlex microsfere è di circa 3 ore, che è molto più veloce del 22 - 24 ore necessario per rivestire il pozzetto di una piastra ELISA, seguita dal trattamentodei pozzetti. Le prestazioni del dosaggio xMAP è anche superiore al ELISA in termini di limite di rilevamento (&lt;4 pg / ml vs&gt; 31 pg / mL) e gamma dinamica (&lt;4 pg / mL a&gt; 8000 pg / ml vs 16 pg / mL a 1000 pg / mL). Lettori di piastre hanno una gamma limitata OD che è 3 o 4 OD, limitando il limite superiore della gamma dinamica per un saggio. Indubbiamente, non tutti gli anticorpi funzionano in un formato ELISA e non tutti gli anticorpi che funzionano bene in un test ELISA sono facilmente trasferibili al formato del saggio xMAP. Tuttavia, poiché i saggi xMAP possono essere multiplati (cioè, funzionare simultaneamente), è possibile valutare diverse cattura e combinazioni anticorpo di rivelazione simultaneamente per identificare il miglior coppia di utilizzare per un saggio. Questo processo consente di risparmiare molto tempo e reagenti rispetto alla procedura ELISA sviluppo, che è limitata alla valutazione di una coppia alla volta. Se due o più coppie di anticorpi di eseguire in modo equivalente; altri parametri del test puòconsiderati per determinare l&#39;idoneità della coppia (ad esempio, la disponibilità, costo, ecc). Oltre a migliorare le prestazioni del dosaggio e la flessibilità con il saggio xMAP, ci sono anche significativi risparmi sui costi. La quantità consigliata di anticorpi necessari per rivestire un singolo pozzo di una piastra ELISA è 400ng, mentre la quantità necessaria per le perline utilizzate in un pozzetto di un saggio xMAP è di circa 7,5 ng. Pertanto, la quantità di anticorpo necessaria per un ELISA e fornirà più di 50 risultati del test, se usato in un saggio xMAP. Per le applicazioni che coinvolgono i campioni preziosi, xMAP ha anche un vantaggio significativo. Il volume di campione raccomandato per l&#39;ELISA è 100 pl, mentre il volume richiesto per il test xMAP può essere metà, o meno. In sintesi, la conversione di un saggio ELISA per la piattaforma Luminex xMAP è semplice, efficiente ed economico, producendo un saggio con una gamma dinamica e sensibilità.

<table border="1"><tbody><tr><td> Descrizione </td><td> Venditore </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Umano TNF-α/TNFSF1A DuoSet kit ELISA </td><td> R &amp; D Systems </td><td> DY210 </td><td> Comprende anticorpi monoclonali accoppiati e biotina policlonali e ricombinante standard di TNF-α proteina </td></tr><tr><td> Anticorpo monoclonale anti TNF-α </td><td> Abcam </td><td> Ab18696 </td><td> Anticorpo di cattura, clone CH8820 </td></tr><tr><td> Anticorpo monoclonale anti TNF-α </td><td> Abcam </td><td> Ab16166 </td><td> Coniugato con biotina Detection, clone AS1 </td></tr><tr><td> TNF alfa proteine </td><td> Abcam </td><td> Ab9642 </td><td> Ricombinante TNF-α proteina standard </td></tr><tr><td> Anticorpo monoclonale anti TNF-α </td><td> NOVUS </td><td> NBP1-50115 </td><td> Anticorpo di cattura, clone 4H31 </td></tr><tr><td> Anticorpo monoclonale anti TNF-α </td><td> Novus </td><td> NB100-78162 </td><td> Coniugato con biotina Detection, clone MAb11 </td></tr><tr><td> TNF alfa proteine </td><td> Novus </td><td> NBC1-18460 </td><td> Ricombinante TNF-α proteina standard </td></tr><tr><td> Anticorpo monoclonale anti TNF-α </td><td> EMD Millipore </td><td> MAB1141 </td><td> Anticorpo di cattura, clonare 3C7.2 </td></tr><tr><td> Anticorpo policlonale di TNF-α </td><td> EMD Millipore </td><td> 654250 </td><td> Anticorpo di coniglio policlonale di rilevamento </td></tr><tr><td> Streptavidina-ficoeritrina </td><td> Muschio </td><td> SAPE-001 </td><td> Giornalista reagente fluorescente per Assay Luminex xMAP </td></tr><tr><td> MAGPIX w / Software Xponent </td><td> Luminex Corporation </td><td> MAGPix-Xponent </td><td> Strumento Luminex </td></tr><tr><td> xMAP anticorpi Coupling (ABC) Kit </td><td> Luminex Corporation </td><td> 40-50016 </td><td> Include reagente EDC, solfo-NHS reattivo, buffer di attivazione, tampone di lavaggio, provette da 1,5 ml di reazione, e pipette monouso </td></tr><tr><td> MagPlex Microsfere, bassa concentrazione </td><td> Luminex Corporation </td><td> MC10012-ID, MC10013-ID, MC10014-ID, MC10015-ID </td><td> Perle di bassa concentrazione (@ 2,5 x 10 6 tallone / mL) </td></tr><tr><td> EZ-Link solfo-NHS-LC-biotina </td><td> Thermo Fisher </td><td> PI-21335 </td><td> Kit biotinilazione per gli anticorpi di rilevazione non modificata </td></tr><tr><td> Tecan Infinite F200 Reader </td><td> Tecan </td><td></td><td> Lettore di piastra ELISA </td></tr><tr><td> Soluzione tampone fosfato </td><td> Sigma-Aldrich </td><td> P-3688 </td><td> 1% BSA-PBS, Assay Buffer </td></tr><tr><td> One-Pint Compact Ultrasonic Cleaner, 115 VAC </td><td> Cole-Parmer </td><td> WU-08849-00 </td><td> Produrre una frequenza effettiva di funzionamento di 55 kHz </td></tr><tr><td> Tubo separatore magnetico </td><td> Luminex Corporation </td><td> CN-0288-01 </td><td> Per singola provetta da 1,5 ml di separazione magnetica in fasi di lavaggio di accoppiamento </td></tr><tr><td> Piastra di separazione magnetica </td><td> Luminex Corporation </td><td> CN-0269-01 </td><td> Per le 96 e piastra di separazione magnetica in fasi di lavaggio del test </td></tr></tbody></table>

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

L&#39;enzima-test immunoenzimatico (ELISA) è stato a lungo il principale strumento per la rilevazione di analiti di interesse in campioni biologici sia per la ricerca delle scienze della vita e la diagnostica clinica. Tuttavia, ELISA ha dei limiti. Esso è tipicamente effettuata in una micropiastra da 96 pozzetti, ed i pozzetti sono rivestite con anticorpo di cattura, che richiede una quantità relativamente grande di campione per catturare un antigene di interesse. L&#39;ampia superficie dei pozzetti e il legame di anticorpo di cattura idrofobo può anche portare a non specifico sfondo legame e maggiore. Inoltre, la maggior parte ELISA invocare mediata da enzimi amplificazione del segnale al fine di ottenere una sensibilità ragionevole. Tale amplificazione non è sempre lineare e può risultati così skew. Negli ultimi 15 anni, una nuova tecnologia è emerso che offre i vantaggi del test ELISA, ma anche permette un throughput più elevato, maggiore flessibilità, volume del campione ridotto e costi inferiori, con un wo similerkflow 1, 2. Luminex xMAP Technology è una (tallone), piattaforma di array di microsfere permettendo sia monoplex e saggi di multiplex che possono essere applicati sia per applicazioni di proteine ​​e acidi nucleici 3-5. Le perle hanno l&#39;anticorpo di cattura immobilizzato covalentemente su una superficie più piccola, richiedendo meno anticorpo di cattura e piccoli volumi di campione, rispetto alla ELISA, e il legame non specifico è significativamente ridotta. Piccoli volumi di campione sono importanti quando si lavora con campioni limite come liquido cerebrospinale, liquido sinoviale, ecc 6. Multiplexing del dosaggio riduce ulteriormente i requisiti di volume del campione, consentendo di raggiungere risultati multipli da un singolo campione. Recenti miglioramenti di Luminex sono: il nuovo sistema MAGPIX, uno più piccolo, meno costoso, più facile da usare analizzatore, a bassa concentrazione magnetica MagPlex Microsfere che eliminano la necessità di costosi e piastre filtranti sono disponibili in una concentrazione di lavoro più adatto per lo sviluppo del test elow-throughput delle applicazioni e l&#39;anticorpo xMAP Coupling (ABC) del kit, che comprende un protocollo, reagenti e materiali di consumo necessari per l&#39;accoppiamento perline per l&#39;anticorpo di cattura di interesse. (Vedere la sezione Materiali per un elenco dettagliato del contenuto del kit.) In questo esperimento, si convertire un pre-ottimizzato saggio ELISA per il TNF-alfa citochina alla piattaforma xMAP e confrontare le prestazioni dei due metodi 7-11. TNF-alfa è un biomarker utilizzato nella misurazione delle risposte infiammatorie in pazienti con disturbi autoimmuni. Cominciamo con l&#39;accoppiamento di anticorpi di cattura quattro candidati a quattro differenti set di microsfere o regioni. Quando miscelati insieme, questi quattro insiemi consentire la prova simultanea di tutti i quattro candidati con quattro anticorpi rilevamento separato per determinare la coppia migliore anticorpo, reagenti risparmio, campione e il tempo. Due saggi xMAP vengono costruiti con le due coppie di anticorpi più ottimali e la loro performance èrispetto a quella originale del saggio ELISA per quanto riguarda la potenza del segnale, gamma dinamica, e la sensibilità.

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

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

Un ELISA può essere facilmente convertito in un saggio Luminex xMAP e, attraverso i benefici di multiplexing, diversi anticorpi possono essere proiettati contemporaneamente per identificare una coppia ottimale anticorpo, con conseguente aumento della sensibilità e gamma dinamica, riducendo i costi dosaggio.

Conversione di un ELISA Capture ad un saggio Luminex xMAP utilizzando un anticorpo Multiplex metodo di screening

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 Views.  Luminex Corporation. Un ELISA può essere facilmente convertito in un saggio Luminex xMAP e, attraverso i benefici di multiplexing, diversi anticorpi possono essere proiettati contemporaneamente per identificare una coppia ottimale anticorpo, con conseguente aumento della sensibilità e gamma dinamica, riducendo i costi dosaggio.

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

Laser Capture Microdissection of Mammalian Tissue

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

Laser Microdissezione Cattura di tessuti di mammiferi

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

Ricerca

Biologia

Cell Capture Using a Microfluidic Device

Cattura cellula Utilizzando un dispositivo a microfluidi

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.

Metodo per la colorazione intero anticorpi Monte a 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.

RNAi di screening per identificare fenotipi postembrionale in 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 di screening a fluorescenza per l&#39;identificazione di modulatori Canali 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 liquido fase di cattura test affinità utilizzando biglie magnetiche per studiare interazioni proteina-proteina: il poliovirus-Nanobody Esempio

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.

High-throughput screening funzionale utilizzando una casalinga 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 ...

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

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

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

Un metodo per la selezione di aptameri-struttura di commutazione Applicato ad una colorimetrico oro nanoparticelle 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.

Identificazione delle coppie Kinase-substrato Usando 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 screening utilizzando un high-throughput genetica 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 ...