Cette recherche a été financée par le Programme de recherche intra-muros de l&#39;NIDCR.

1. Aperçu schématique: Cette vidéo montre les étapes nécessaires à réaliser le dosage LIPS. Les antigènes sont exprimés dans les cellules COS1 que la luciférase de Renilla recombinante (RUC)-antigène fusions, et des extraits bruts sont obtenus et utilisés sans purification. Le dosage de LIPS est initiée par l&#39;incubation crud e Ruc-antigène des extraits avec des sérums de patients dans les puits de microtitration. Le mélange antigène-anticorps est ensuite transférée à une plaque de 96 puits filtre contenant la protéine A / G perles pour capturer les molécules d&#39;IgG. Après avoir lavé le filtre à plaques contenant la protéine, un anticorps / G perles, liée Ruc-antigène est mesurée par l&#39;addition du substrat coelentérazine et les unités de la lumière sont mesurées avec un luminomètre. PARTIE 1: La transfection de plasmides pour la production de protéines de fusion Renilla-antigène Set-up: COS1 cellules sont cultivées dans du DMEM-FCS 10% en utilisant des protocoles standard de culture de tissus. Plasmides pour Renilla luciférase fusions ont été décrits précédemment 1. ADN de ces plasmides est préparé en utilisant un kit de Qiagen Midiprep. Le rendement devrait être d&#39;environ 1 mg -3. Mesurer la concentration d&#39;ADN et de stocker en tant que mg 1000 / ml de solution concentrée à -20 ° C. Procédure: <ol><li> Un jour avant la transfection Cos-1, diviser les cellules en nouvelle 100 x 20 mm plats à environ 2 x 10 6 par plaque et incuber à 37 ° C. </li><li> Le jour suivant, les cellules COS-1 devrait être de 80-95% confluentes. Étiquette tubes de 1,5 ml de microcentrifugation en polypropylène pour chaque ADN plasmidique à transfecter. Laisser le réactif de transfection FuGENE-6, qui est stocké à 4 ° C, se réchauffer à température ambiante. </li><li> Ajouter 94 ul d&#39;Opti-MEM médias pour chaque tube de centrifugeuse. Ensuite, ajoutez 6 pl d&#39;FuGENE 6 aux médias Opti-MEM sans toucher la paroi latérale. </li><li> Incuber le mélange pendant 5 minutes à température ambiante. </li><li> Ajouter 1-2 ug (en stock ADN 1mg/ml) du plasmide de fusion antigène Renilla luciférase construire. Mélanger et incuber le mélange pendant 15 minutes à température ambiante. </li><li> Transférer la solution d&#39;ADN-FuGENE 6 Opti-MEM pour les cellules en la goutte uniformément dans les médias de l&#39;COS1 cellules. </li></ol> PARTIE 2: Récolte Renilla-antigène Fusions <ol><li> Deux jours après transfection, les cellules COS-1 sont récoltées. Ceci est initiée par la suppression des médias et puis à rincer les cellules avec 6 ml de PBS. Après décantation du PBS, une pipette de suite tout résidu PBS de la boîte de culture tissulaire. </li><li> Ajouter 1,4 ml de tampon de lyse froid composé de 50 mM Tris, pH 7,5, NaCl 100 mM, MgCl2 5 mM, 1% de Triton X-100, glycérol 50%, et les inhibiteurs de protéase (2 comprimés de cocktail miniprotease inhibiteur de terminer par 50 ml de tampon de lyse). Récolte des cellules avec un racloir cellulaire et transférer rapidement la moitié de la lysat à chacun des deux tubes de 1,5 ml de microcentrifugation sur la glace. </li><li> Un Sonifier Branson 150 est utilisée pour briser les cellules ouvertes. Placer le microtube contenant le lysat cellulaire sur la glace et le pouls pendant 5 sec, 5 sec et 5 sec avec les réglages sonication de 2, 2 et 4, respectivement. </li><li> Centrifuger le lysat cellulaire à 12 500 RPM pour deux rotations 4 minute à 4 ° C. Après la première rotation, retourner doucement les tubes pour éliminer les débris lâchement de la paroi latérale du tube. Après la seconde vrille, transférer soigneusement le surnageant, sans perturber le culot, à partir de deux tubes d&#39;un tube de micro nouvelles sur la glace. </li><li> Calculer les unités légères (UGB) par de lysat. Pour mesurer la LU, diluer 1 de lysat avec 8 pi de PBS dans un tube de centrifugeuse de nouvelles. Directement ajouter 100 ul de substrat 1X coelentérazine au mélange dilué et mesurer immédiatement la luminescence dans le tube en utilisant un luminomètre tubes (20/20 n Turner scientifique) avec une lecture de 5 secondes. </li><li> Stocker le lysat Ruc-antigène à -20 ° C pendant 1-2 jours ou stocker pour une longue période de temps en aliquots à -80 ° C. </li></ol> PARTIE 3: Préparer une plaque de Sera maître <ol><li> Faites une plaque maître sérums en ajoutant d&#39;abord 450 pi de tampon A (Tris 50 mM, pH 7,5, NaCl 100 mM, MgCl2 5 mM, 1% Triton X-100) dans chaque puits d&#39;une plaque de 96 puits profonds polypropylène microtitration . À cette étape, un colorant, rouge de phénol, peuvent également être inclus dans le tampon A (concentration finale est de 0,2 ug / ml dans le tampon A) pour agir comme un traceur pour la surveillance future ajout de l&#39;échantillon des sérums et autres étapes de l&#39;essai LIPS. </li><li> Ensuite, ajoutez 50 pl de sérum de chaque échantillon dans les puits différents contenant 450 pi de tampon A. Notez que ceci est une dilution 1:10 de l&#39;sérum dans le tampon A. Typiquement sérums ne sont pas ajoutées aux deux derniers puits de la plaque maître car ceci est réservé pour les blancs de mémoire tampon. </li><li> Avant d&#39;utiliser la plaque de maître, il est largement ébranlée (1-2 heures) sur une plate-forme des rotateurs. Le sérum de la plaque maître est stable pour au moins 1 mois (ou plus) à 4 ° C, si stocké correctement pour éviter l&#39;évaporation. </li><li> Comme décrit ci-dessous, cette plaque maître fournit 10 l de sérum dilué à plusieurs reprises enlevé pour le profilage des sérums dirigés contre les antigènes multiples. Plaques maîtres grands et petits peuvent aussi être employées. Sceller la plaque bien avec Parafilm quand il n&#39;est pas utilisé. </li></ol> PARTIE 4: dosage de LIPS <ol><li> Polypropylène 96-superficielle des plaques de microtitration et sont utilisés pour tester les sérums. Dans la première étape, ajouter 40 ul de tampon A dans chaque puits de la plaque de 96 puits en utilisant une micropipette à 8 canaux. </li><li> Prenez ensuite 10 ul de dilution des sérums (1 équivalent ul sérums) de la plaque de maître et de l&#39;ajouter directement à chaque puits de la plaque de travail en utilisant une micropipette à 8 canaux. </li><li> Un mélange maître contenant l&#39;extrait de Ruc-antigène est la prochaine formulé tel que 1 x 10 7 unités de lumière (LU) est ajouté dans 50 ul de tampon A dans chaque puits. Basse entrées (aussi peu que 1 x 10 6) peut également être utilisé, mais entraîner une baisse de plage dynamique. Faire ce mélange maître et la pipette 50 ul d&#39;Ruc-antigène mélange à chaque puits. </li><li> Incuber la plaque sur un agitateur rotatif pendant 1 heure à température ambiante. </li><li> Durant l&#39;incubation, ajouter 5 pi d&#39;une suspension de 30% de protéines Ultralink A / G perles (Pierce Biotechnology, Rockford, IL) dans le PBS au fond de chaque puits d&#39;une nouvelle plaque filtrante 96 puits HTS (Millipore, Bedford, MA) . </li><li> Après l&#39;incubation de 1 heure, le transfert des 100 ul Ruc-antigène des anticorps mélange réactionnel plaques à 96 puits HTS filtre contenant la protéine A / G perles en utilisant une micropipette à 8 canaux. </li><li> Incuber la plaque de 96 puits de filtre sur un agitateur rotatif pendant 1 heure supplémentaire à température ambiante. </li><li> Laver ensuite la plaque de filtre sur une rampe à vide. Chaque puits est lavé 8 fois avec 100 pi de tampon A, suivie par deux fois avec 100 ul de PBS. Ceci peut être réalisé manuellement ou avec une station de pipetage robotique. </li><li> Après le dernier lavage, le vide est éteint. Retirer la plaque de filtration et de tamponner sec en utilisant une pile de serviettes en papier ou papier filtre en veillant à éliminer l&#39;humidité sur le haut et le bas de la plaque. </li></ol> PARTIE 5: Mesure de l&#39;analyse des données et de luminescence Set-up: Un LB Berthold microplaques 960 Centro luminomètre est utilisé pour déterminer la luminescence dans chaque puits en utilisant un seul injecteur. Une fois que la machine est en marche, rincez à l&#39;eau distillée de l&#39;injecteur H 2 O en utilisant le cycle de lavage injecteur. Substrat coelentérazine est préparé en utilisant le kit Promega substrat Renilla comme décrit par le fabricant. Typiquement 6 ml de mélange de substrat coelentérazine 1X (soit 60 actions coelentérazine ul + 6 ml de tampon 1X) est préparé pour l&#39;amorçage de la machine et exécutant l&#39;un assiette pleine de 96 puits. Avant d&#39;analyser la plaque, le LB 960 Berthold microplaques Centro luminomètre est amorcée avec 1X substrat coelentérazine. Ouvrir un fichier contenant la configuration du programme pour l&#39;injection du substrat et de la lecture de la plaque. Pour ces mesures, 50 ul de substrat coelentérazine est injecté, la plaque est agitée pendant 2 secondes, suivie par une seconde lecture de la luminescence 5. <ol><li> Bien que le luminomètre plaque a la capacité de lire la plaque entière, une lecture partielle de la plaque peut également être sélectionné (sous le menu lecture). Démarrez le programme, ce qui déclenche la lecture de la plaque. </li><li> Après la course, enlever la plaque filtrante de microtitration rapidement pour éviter tout déversement dans le luminomètre. Exporter les données générées avec le programme MikroWin dans un format Excel pour l&#39;analyse. </li><li> Nous vous recommandons d&#39;évaluer les sérums des deux manches indépendant. Les données sont ensuite moyennées pour générer les valeurs titre LU pour chaque échantillon. Ces valeurs peuvent encore être ajusté pour soustraire les blancs de mémoire tampon. </li><li> Complémentaires d&#39;analyse de données telles que la détermination de la coupure peut être calculée en prenant la moyenne plus de 3 ou 5 écarts-types des valeurs de contrôle. </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>

LIPS nécessite l&#39;optimisation des essais minime et, en raison de sa simplicité, les données de haute qualité peuvent généralement être générés en utilisant les lèvres en moins de deux semaines pour tout antigène donné. Les étapes les plus consommatrices de temps sont le clonage et la génération du vecteur d&#39;expression approprié plasmide contenant la fusion Ruc-antigène. Une fois ces plasmides sont générés, les antigènes simples ou multiples peuvent être rapidement testé avec les lèvres comme décrit ci-dessus. Il est à noter que les lèvres est assez polyvalent tels que les sérums peuvent également être testés dans un format tube ou encore sur une plaque de microtitration avec filtre à l&#39;aide d&#39;un poste de travail robotique Biomek 2 ou un laveur de plaques de filtration sous vide. Ce système hautement évolutif permet l&#39;lèvres parallèles profilage d&#39;un panel d&#39;autoantigènes humaine 3 ou le protéome entier d&#39;un quatre virus. Il est probable que de nombreux profils d&#39;anticorps différents générés par LIPS sera utile pour le diagnostic de 3-9, antigène de la découverte 9, suivant le cours d&#39;un traitement de 10, la surveillance des vaccins et a de larges implications pour des maladies spécifiques et pour la santé publique en général.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>
		</table>
		</div>

antibody profiling by luciferase immunoprecipitation systems (lips)

Technologies pour complètement comprendre et quantifier les profils anticorps à des auto-antigènes et les agents infectieux peuvent produire de nouvelles connaissances sur les mécanismes de la maladie et peut éclairer de nouveaux marqueurs à substratify maladie avec différentes caractéristiques cliniques et de mieux comprendre la pathogenèse. Nous avons développé une méthode hautement quantitative appelés systèmes immunoprécipitation luciférase (LIPS) pour le profilage des patients réponses anticorps sérum à des autoantigènes et les antigènes des pathogènes associés à l&#39;infection. Contrairement ELISA, les lèvres très sensibles est facilement mis en œuvre pour l&#39;enquête humorale profils de réponse sérologique aux antigènes différents dans un format universel et produit le titre d&#39;anticorps dynamique des distances allant jusqu&#39;à 6 log<sub&gt; 10</sub&gt; Pour certains antigènes. Dans ces études, le profilage quantitatives par des lèvres du patient réponses humorale contre les antigènes des panneaux ou même l&#39;ensemble du protéome de certains agents pathogènes (ie VIH), est généralement plus informative que les tests un seul antigène par ELISA. En outre, LIPS élimine également le temps et les efforts nécessaires pour produire des antigènes hautement purifiés ainsi que les étapes de main-d&#39;œuvre nécessaire à l&#39;optimisation des essais ELISA standard. Ici, nous fournir un protocole détaillé décrivant les aspects techniques de l&#39;exécution des tests pour LIPS facilement profilage réponses des anticorps à des antigènes uniques ou multiples.

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

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

Les aspects techniques du spectacle LIPS (Systèmes immunoprécipitation luciférase) sont décrits. L&#39;approche globale consiste à exprimer des gènes codant pour des antigènes chimériques fusionnées à luciférase de Renilla (RUC) dans les cellules de mammifères. Ruc-antigène brut extraits sont ensuite préparés et, sans purification, employées dans des essais d&#39;immunoprécipitation de quantifier les anticorps.

Profilage anticorps par immunoprécipitation Systèmes luciférase (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 vues.  National Institute of Dental and Craniofacial Research, National Institutes of Health. Les aspects techniques du spectacle LIPS (Systèmes immunoprécipitation luciférase) sont décrits. L'approche globale consiste à exprimer des gènes codant pour des antigènes chimériques fusionnées à luciférase de Renilla (RUC) dans les cellules de mammifères. Ruc-antigène brut extraits sont ensuite préparés et, sans purification, employées dans des essais d'immunoprécipitation de quantifier les anticorps.

Vidéo: 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.

Phénomènes AC électrocinétique générées par des structures microélectrodes

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, ...

Recherche

Biologie

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).

Immunoprécipitation ADN méthylé

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: immunoprécipitation détectée par cytométrie en flux

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.

Transcription in vitro et plafonnement de l&#39;ARNm de luciférase Gaussia Suivie par la transfection de cellules HeLa

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.

Microarray Anticorps chimiquement bloqué pour multiplexée à haut débit de profilage de glycosylation des protéines spécifiques dans les échantillons complexes

 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.

Induction et analyse des épithéliale à mésenchymateuses transition

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.

Analyse de la traduction Initiation Pendant conditions de stress par polysome profilage

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.

Profilage individuel embryonnaires humaines Cellules souches par RT-PCR quantitative

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.

Évaluation de la sélective ARNm traduction dans les cellules de mammifères par polysome profilage

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.

Fecal glucocorticoïdes Analyse: Surveillance surrénale non invasive en Equidés

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