Questa ricerca è stata sostenuta dal Programma di ricerca intramurale del NIDCR.

1. Panoramica schematica: Questo video mostra i passaggi necessari per eseguire il test labbra. Gli antigeni sono espressi in Cos1 cellule come luciferasi ricombinante Renilla (Ruc)-antigene fusioni, e gli estratti grezzi sono ottenuti e utilizzati senza purificazione. Il test LABBRA è avviata da incubando crud e Ruc-antigene estratti con sieri dei pazienti in pozzetti. L&#39;anticorpo-antigene miscela viene poi trasferito su una piastra a 96 pozzetti filtro contenente la proteina A / G perline per catturare molecole di IgG. Dopo aver lavato la piastra filtro contenente la proteina Un anticorpo / G perline, legato Ruc-antigene è misurato con l&#39;aggiunta di substrato coelenterazine e le unità di luce sono misurati con un luminometro. PARTE 1: La trasfezione di plasmidi per la produzione di Renilla Antigen-proteine ​​di fusione Set-up: Cos1 le cellule sono coltivate in DMEM-10% FCS usando i protocolli standard di coltura dei tessuti. Plasmidi per Renilla fusioni luciferasi sono stati descritti in precedenza 1. DNA per questi plasmidi è preparata con un kit Midiprep da Qiagen. Il rendimento dovrebbe essere di circa 1 mg -3. Misurare la concentrazione di DNA e conservarlo come mcg 1000 / magazzino ml di soluzione a -20 ° C. Procedimento: <ol><li> Un giorno prima Cos-1 transfezione, dividere le celle in nuovi piatti 100 x 20 mm a circa 2 x 10 6 per piastra e incubare a 37 ° C. </li><li> Il giorno seguente, il Cos-1 le cellule dovrebbero essere 80-95% confluenti. Etichetta 1,5 ml tubi in polipropilene microcentrifuga per ogni DNA plasmidico da transfettate. Lasciare che il FuGENE-6 reagenti di trasfezione, che viene conservato a 4 ° C, per riscaldare a temperatura ambiente. </li><li> Aggiungere 94 ml di Opti-MEM media per ogni provetta per microcentrifuga. Prossimo aggiungere 6 ml di FuGENE 6 al Opti-MEM multimediali senza toccare la parete laterale. </li><li> Incubare la miscela per 5 minuti a temperatura ambiente. </li><li> Aggiungere 1-2 mcg (da stock DNA 1mg/ml) di plasmide per Renilla fusione antigene luciferasi costruire. Miscelare e incubare la miscela per 15 minuti a temperatura ambiente. </li><li> Trasferire il DNA FuGENE soluzione 6-Opti-MEM per le cellule aggiungendo qualche goccia in modo uniforme nei media del Cos1 cellule. </li></ol> PARTE 2: raccolta Renilla antigene-Fusioni <ol><li> Due giorni dopo la transfezione, il Cos-1 le cellule vengono raccolte. Questo è iniziata rimuovendo il materiale e poi risciacquare le cellule con 6 ml di PBS. Dopo la decantazione del PBS, pipetta via qualsiasi residuo di PBS dal piatto coltura di tessuti. </li><li> Aggiungere 1,4 ml di tampone di lisi freddo composto da 50 mM Tris, pH 7,5, 100 mM NaCl, 5 mM MgCl 2, 1% Triton X-100, glicerolo 50% e inibitori della proteasi (2 compresse di cocktail completo inibitore miniprotease per 50 ml di tampone di lisi). Celle di raccolta con un scrapper cellula e trasferire rapidamente la metà del lisato a ciascuno dei due tubi da 1,5 ml microcentrifuga sul ghiaccio. </li><li> Un Sonifier Branson 150 è utilizzata per rompere le cellule aperte. Collocare la provetta contenente il lisato cellulare su ghiaccio e di impulsi per 5 secondi, 5 secondi e 5 secondi con le impostazioni di sonicazione di 2, 2 e 4, rispettivamente. </li><li> Centrifugare il lisato cellulare a 12.500 RPM per due 4 giri minuto a 4 ° C. Dopo il primo giro, capovolgere delicatamente i tubi per rimuovere i detriti liberamente attaccata dalla parete laterale del tubo. Dopo il secondo giro, trasferire con cautela il surnatante, senza disturbare il pellet, dai due tubi di una provetta per microcentrifuga nuovo sul ghiaccio. </li><li> Calcolare i gruppi ottici (UBA) per ml di lisato. Per misurare la LU, diluire 1 ml di lisato con 8 ml di PBS in una provetta per microcentrifuga nuovo. Direttamente aggiungere 100 ml di 1X substrato coelenterazine alla miscela diluita e misurare immediatamente luminescenza nel tubo con un luminometro tubo (20/20 n Turner scientifico) con una lettura 5 secondi. </li><li> Conservare la Ruc-antigene lisato a -20 ° C per 1-2 giorni o conservare per un periodo più lungo di volte in aliquote a -80 ° C. </li></ol> PARTE 3: Preparazione di una piastra Sera master <ol><li> Fai una lastra sieri in primo luogo l&#39;aggiunta di 450 ml di tampone A (50 mM Tris, pH 7,5, 100 mM NaCl, 5 mM MgCl 2, 1% Triton X-100) in ciascun pozzetto di una 96-profondo-pozzetti in polipropilene microtitolazione . A questo punto, un colorante, rosso fenolo, può anche essere incluso in un tampone A (concentrazione finale è di 0,2 mg / ml in tampone A) di agire come tracciante per il monitoraggio futuro l&#39;aggiunta del campione sieri e altri passi del test labbra. </li><li> Prossimo aggiungere 50 ml di siero di ogni campione nei pozzetti differenti contenente 450 ml di tampone A. Si noti che questa è una diluizione 1:10 del siero in tampone A. Tipicamente sieri non viene aggiunto gli ultimi due pozzetti della piastra maestro perché questo è riservato per i campi del buffer. </li><li> Prima di utilizzare il piatto principale, è ampiamente scossa (1-2 ore) su una piattaforma dei rotatori. Il siero nella piastra principale è staBLE per almeno 1 mese (o più) a 4 ° C, se conservato correttamente per evitare l&#39;evaporazione. </li><li> Come descritto più avanti, questa piastra master fornisce 10 ml di siero diluito per essere ripetutamente rimosso per la profilatura dei sieri contro antigeni multipli. Piastre maestro grandi e piccole possono anche essere impiegati. Sigillare la piastra bene con Parafilm quando non viene utilizzato. </li></ol> PARTE 4: analisi LIPS <ol><li> Polipropilene 96-superficiale micropiastre e sono usati per testare i sieri. Nella prima fase, aggiungere 40 ml di tampone A in ciascun pozzetto della piastra a 96 pozzetti utilizzando una micropipetta 8 canali. </li><li> Prossimo prendere 10 ml di siero diluito (1 equivalente microlitri sieri) dalla piastra master e aggiungerlo direttamente a ciascun pozzetto della piastra di lavoro con un 8 micropipetta canale. </li><li> Un master mix contenente i Ruc-antigene estratto è accanto formulata in modo tale che 1 x 10 7 gruppi ottici (LU) si aggiunge in 50 ml di tampone A per ciascun pozzetto. Basso input (anche solo di 1 X 10 6) può essere utilizzato anche, ma si ottiene una gamma dinamica più bassa. Rendono questa miscela maestro e pipettare 50 ml di Ruc-antigene miscela in ciascun pozzetto. </li><li> Incubare la piastra su un agitatore rotante per 1 ora a temperatura ambiente. </li><li> Durante l&#39;incubazione, aggiungere 5 ml di una sospensione al 30% di proteine ​​Ultralink A / G perline (Pierce Biotecnologie, Rockford, IL) in PBS al fondo di ogni pozzetto di un nuovo 96 pozzetti filtro HTS piastra (Millipore, Bedford, MA) . </li><li> Dopo l&#39;incubazione 1 ora, trasferire il 100 ul Ruc miscela di reazione antigene-anticorpo a 96 e filtro piastre HTS contenente la proteina A / G con perline micropipetta a 8 canali. </li><li> Incubare la piastra a 96 pozzetti filtro su un agitatore rotante per 1 ora in più a temperatura ambiente. </li><li> Lavare quindi la piastra di filtro su un collettore di aspirazione. Ogni pozzetto viene lavato 8 volte con 100 ml di tampone A, seguito da due volte con 100 ml di PBS. Questa operazione può essere effettuata manualmente o con una workstation pipettaggio robotica. </li><li> Dopo l&#39;ultimo lavaggio, il vuoto è spento. Rimuovere la piastra di filtro e asciugare asciugare con una pila di tovaglioli di carta o carta da filtro avendo cura di rimuovere l&#39;umidità nella parte superiore e inferiore della piastra. </li></ol> PARTE 5: misura e analisi dei dati Luminescenza Set-up: Un Berthold LB 960 Centro micropiastre luminometro è utilizzato per determinare luminescenza in ciascun pozzetto utilizzando un singolo iniettore. Una volta che la macchina è accesa, sciacquare con acqua distillata l&#39;iniettore H 2 O mediante il ciclo di lavaggio iniettori. Substrato Coelenterazine è preparato con il kit Renilla Promega substrato, come descritto dal produttore. Tipicamente 6 ml di mix 1X substrato coelenterazine (ovvero 60 magazzino coelenterazine microlitri oltre 6 ml di tampone 1X) è pronto per l&#39;innesco della macchina e che esegue uno piatto pieno a 96 pozzetti. Prima di analizzare la piastra, il Berthold LB 960 Centro micropiastre luminometro è innescato con 1X substrato coelenterazine. Aprire un file di programma che contiene le impostazioni per l&#39;iniezione del substrato e leggere la piastra. Per queste misurazioni, 50 ml di substrato coelenterazine viene iniettato, il piatto è scossa per 2 secondi, seguito da un 5 sec in lettura di luminescenza. <ol><li> Anche se il luminometro piastra ha la capacità di leggere l&#39;intera piastra, una lettura parziale della piastra può essere selezionata (nel menu di lettura). Avviare il programma, che inizia la lettura della piastra. </li><li> Dopo l&#39;esecuzione, rimuovere la piastra microtiter filtro tempestivamente per evitare la fuoriuscita del luminometro. Esportare i dati generati con il programma MikroWin in un formato Excel per l&#39;analisi. </li><li> Si consiglia di valutare i sieri di due piste indipendenti. I dati vengono poi media per generare i valori titolo LU per ogni campione. Questi valori possono essere ulteriormente regolata per sottrarre gli spazi vuoti buffer. </li><li> Ulteriori analisi dei dati, come la determinazione del taglio può essere calcolato prendendo la media più 3 o 5 deviazioni standard dei valori di controllo. </li></ol>

<ol>
	<li>Burbelo, P. D., Goldman, R., Mattson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+immunoprecipitation+method+for+quantitatively+measuring+antibody+responses+in+clinical+sera+samples+by+using+mammalian-produced+Renilla+luciferase-antigen+fusion+proteins.">A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.</a> BMC Biotechnol. 5, 22-22 (2005).</li><li>Burbelo, P. D., Leahy, H. P., Iadarola, M. J., Nutman, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Four-Antigen+Mixture+for+Rapid+Assessment+of+Onchocerca+volvulus+Infection.">A Four-Antigen Mixture for Rapid Assessment of Onchocerca volvulus Infection.</a> PLoS Negl Trop Dis. 3, e438-e438 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+robust+luminescent+profiling+of+anti-La+and+other+autoantibodies+in+Sj+gren&#x2019;s+syndrome.">Sensitive and robust luminescent profiling of anti-La and other autoantibodies in Sj gren&#x2019;s syndrome.</a> Autoimmunity. 139, 241-245 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+antibody+quantification+and+generation+of+whole+proteome+antibody+response+profiles+using+LIPS+(luciferase+immunoprecipitation+systems).">Rapid antibody quantification and generation of whole proteome antibody response profiles using LIPS (luciferase immunoprecipitation systems).</a> Biochem Biophys Res Commun. 352, 889-895 (2007).</li><li>Burbelo, P. D., Groot, S., Dalakas, M. C., Iadarola, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+definition+profiling+of+autoantibodies+to+glutamic+acid+decarboxylases+GAD65/GAD67+in+stiff-person+syndrome.">High definition profiling of autoantibodies to glutamic acid decarboxylases GAD65/GAD67 in stiff-person syndrome.</a> Biochem Biophys Res Commun. 366, 1-7 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+luminescence+assay+for+autoantibodies+to+mammalian+cell-prepared+insulinoma-associated+protein+2.">A new luminescence assay for autoantibodies to mammalian cell-prepared insulinoma-associated protein 2.</a> Diabetes Care. 31, 1824-1826 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+diagnosis+of+human+herpes+simplex+virus+type+1+and+2+infections+by+luciferase+immunoprecipitation+system+assay.">Serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system assay.</a> Clin Vaccine Immunol. 16, 366-371 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+quantitative+serological+detection+of+anti-cytomegalovirus+(CMV)+antibodies.">Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies.</a> Virol J. 6, 45-45 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-antigen+mixture+containing+v-cyclin+for+serological+screening+of+human+herpesvirus+8+infection.">Four-antigen mixture containing v-cyclin for serological screening of human herpesvirus 8 infection.</a> Clin Vaccine Immunol. 16, 621-627 (2009).</li><li>Ramanathan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+luciferase+immunoprecipitation+systems+assay+enhances+the+sensitivity+and+specificity+of+diagnosis+of+Strongyloides+stercoralis+infection.">A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection.</a> J Infect Dis. 198, 444-451 (2008).</li></ol>

LABBRA richiede l&#39;ottimizzazione minimo dosaggio e, grazie alla sua semplicità, dati di alta qualità in genere può essere generato usando LIPS in meno di due settimane per un dato antigene. I passi più tempo sono la clonazione e generazione del vettore di espressione appropriato plasmide contenente il Ruc antigene-fusione. Una volta che questi plasmidi sono generati, antigeni singole o multiple possono essere rapidamente testato con LABBRA come descritto sopra. Va notato che le labbra è molto versatile, in modo tale che i sieri possono essere testati in un formato tubo o in alternativa su una piastra di microtitolazione filtro con l&#39;ausilio di una workstation robotica Biomek 2 o una rondella piatto con filtrazione a vuoto. Questo sistema altamente scalabile permette di LABBRA parallelo profilazione di un panel di umano autoantigeni 3 o l&#39;intero proteoma di un 4 virus. E &#39;probabile che molti profili diversi anticorpi generati dalla LABBRA sarà utile per la diagnostica 3-9, antigene scoperta 9, seguendo il corso di un trattamento di 10, il monitoraggio del vaccino e ha vaste implicazioni per stati patologici specifici e per la salute pubblica in generale.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HiSpeed Plasmid Midi KIt</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
<tr>
<td>100 x 20 mm tissue culture dishes</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>353003</td>
<td>&nbsp;</td>
<tr>
<td>Opti-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>FuGENE 6</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1814443 </td>
<td>&nbsp;</td>
<tr>
<td>Complete, Mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836153001</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 96 DeepWell 1 ml plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120 </td>
<td>Deep well plate with lid and seal with Parafilm</td>
</tr>
<tr>
<td>Polypropylene microtiter plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120</td>
<td>&nbsp;</td>
<tr>
<td>HTS Filter plate</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSBVN1B50</td>
<td>&nbsp;</td>
<tr>
<td>Ultralink Immobilized Protein A/G</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>53133 (10ml)</td>
<td>&nbsp;</td>
<tr>
<td>Renilla Luciferase Assay System</td>
<td>&nbsp;</td>
<td>Promega </td>
<td>E2820</td>
<td>For 2000 assays</td>
</tr>
<tr>
<td>Centro microplate luminometer</td>
<td>&nbsp;</td>
<td>Berthold</td>
<td>LB 960</td>
<td>&nbsp;</td>		</tbody>
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antibody profiling by luciferase immunoprecipitation systems (lips)

Tecnologie per la comprensione e la quantificazione completo profili anticorpi autoantigeni e agenti infettivi possono produrre nuove conoscenze sui meccanismi della malattia e può spiegare nuovi marcatori per substratify malattia con differenti caratteristiche cliniche e capire meglio la patogenesi. Abbiamo sviluppato un metodo altamente quantitativo chiamato Sistemi Immunoprecipitazione luciferasi (LIPS) per il profiling dei pazienti la risposta anticorpale sieri di autoantigeni e antigeni patogeni associati alle infezioni. A differenza di ELISA, le labbra molto sensibile può essere facilmente implementato per sondaggio umorale profili risposta sierologica di antigeni diversi in un formato universale e produce titolo anticorpale dinamica campi fino a 6 log<sub&gt; 10</sub&gt; Per alcuni antigeni. In questi studi, profili quantitativi con LABBRA del paziente risposte umorali contro i pannelli di antigeni o addirittura l&#39;intero proteoma di alcuni agenti patogeni (ad esempio l&#39;HIV), è in genere più informativa di test di un singolo antigene di ELISA. Inoltre, LABBRA elimina anche il tempo e lo sforzo necessario per la produzione di antigeni altamente purificati e alta intensità di lavoro passi necessari per l&#39;ottimizzazione del test ELISA standard. Qui forniamo un protocollo dettagliato che descrive gli aspetti tecnici di eseguire test per LABBRA prontamente profiling risposta anticorpale agli antigeni singoli o multipli.

Watch this Scientific Journal Video about Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS at JoVE.com

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

Gli aspetti tecnici di eseguire LIPS (Sistemi Immunoprecipitazione luciferasi) sono descritti. L&#39;approccio generale prevede che esprimono i geni chimerici codifica antigeni fusi a Renilla luciferasi (Ruc) in cellule di mammifero. Greggio Ruc-antigene estratti vengono poi preparati e, senza purificazione, impiegati nei test di immunoprecipitazione per quantificare gli anticorpi.

Profiling anticorpi Systems Immunoprecipitazione luciferasi (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

antibody-profiling-by-luciferase-immunoprecipitation-systems-lips

Research

JoVE Journal

Biology

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

26.7K Views.  National Institute of Dental and Craniofacial Research, National Institutes of Health. Gli aspetti tecnici di eseguire LIPS (Sistemi Immunoprecipitazione luciferasi) sono descritti. L'approccio generale prevede che esprimono i geni chimerici codifica antigeni fusi a Renilla luciferasi (Ruc) in cellule di mammifero. Greggio Ruc-antigene estratti vengono poi preparati e, senza purificazione, impiegati nei test di immunoprecipitazione per quantificare gli anticorpi.

Video: Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

Fenomeni AC elettrocinetico Generato da strutture microelettrodo

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

Ricerca

Biologia

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Immunoprecipitazione DNA metilato

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP-FCM: Immunoprecipitazione rilevato mediante citometria di flusso

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Trascrizione in vitro e tappatura mRNA Luciferase Gaussia Seguito da cellule HeLa Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine ​​specifico in campioni complessi

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Analisi della traduzione innesco durante condizioni di stress da polisoma Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Profiling cellule staminali embrionali umane individuale mediante RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Valutazione della Selective mRNA Traduzione in cellule di mammifero per polisoma Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecale Analisi glucocorticoidi: non invasivo di monitoraggio surrenale in equidi

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...