Diese Arbeit wurde durch die Interne Research Program der NIDCR unterstützt.

1. Schematische Übersicht: Dieses Video zeigt die Schritte bei der Durchführung der LIPS-Test beteiligt. Antigene sind in COS1-Zellen als rekombinante Renilla-Luciferase (RUC)-Antigen Fusionen und Rohextrakte ausgedrückt erhalten und ohne Reinigung verwendet. Die LIPS-Test wird durch Inkubation crud e Ruc-Antigen-Extrakte mit Patientenseren in Mikrotitervertiefungen eingeleitet. Die Antikörper-Antigen-Gemisch wird dann zu einer 96-well Filterplatte mit Protein A / G Perlen IgG-Moleküle einzufangen übertragen. Nach dem Waschen der Filter-Platte mit dem Protein A / G Perlen, Antikörper gebunden Ruc-Antigen, das durch die Zugabe von Coelenterazin Substrat und Licht-Einheiten gemessen werden mit einem Luminometer gemessen. TEIL 1: Transfektion von Plasmiden für die Herstellung von Renilla-Antigen-Fusionsproteine Set-up: COS1 Zellen werden in DMEM-10% FCS mit Standard-Zellkultur-Protokolle kultiviert. Plasmide für Renilla Luziferase Fusionen wurden bereits 1 beschrieben. DNA für diese Plasmide wird unter Verwendung eines Midiprep Kit von Qiagen. Die Ausbeute sollte etwa 1 -3 mg werden. Messen Sie die DNA-Konzentration und speichern als 1000 ug / ml Stammlösung bei -20 ° C. Vorgehensweise: <ol><li> Einen Tag vor der Transfektion aufgeteilt Cos-1 Zellen in neue 100 x 20 mm Gerichte bei ca. 2 x 10 6 pro Platte und bei 37 ° C. </li><li> Am folgenden Tag sollte der Cos-1 Zellen werden 80-95% konfluent. Label 1,5 ml Polypropylen Mikrozentrifugenröhrchen für jedes Plasmid-DNA transfiziert werden. Lassen Sie die FuGENE-6 Transfektionsreagenz, welches bei 4 ° C gelagert, um sich aufzuwärmen, um Raumtemp. </li><li> Add 94 ul der Opti-MEM Medien in jedes Mikrozentrifugenröhrchen. Als nächstes fügen Sie 6 ul FuGENE 6 zur Opti-MEM Medium ohne Berührung der Seitenwand. </li><li> Inkubieren Sie die Mischung für 5 Minuten bei Raumtemperatur. </li><li> Fügen Sie 1-2 pg (von 1mg/ml DNA stock) von Plasmid für Renilla Luziferase Antigen Fusionskonstrukt. Mix und dann inkubieren die Mischung für 15 Minuten bei Raumtemperatur. </li><li> Übertragen Sie die DNA-FuGENE 6-Opti-MEM-Lösung, um die Zellen durch Abtropfen es gleichmäßig in den Medien der COS1 Zellen. </li></ol> TEIL 2: Die Ernte Renilla-Antigen-Fusions <ol><li> Zwei Tage nach der Transfektion werden die Cos-1 Zellen geerntet. Dies wird durch Entfernen des Mediums und dann Spülen der Zellen mit 6 ml PBS eingeleitet. Nach Dekantieren des PBS, Pipette entfernt restliche PBS aus dem Gewebe Kulturschale. </li><li> Fügen Sie 1,4 ml kaltem Lysepuffer 50 mM Tris, pH 7,5, 100 mM NaCl, 5 mM MgCl 2, 1% Triton X-100, 50% Glycerin und Protease-Inhibitoren (2 Tabletten von kompletten miniprotease Inhibitor Cocktail pro 50 ml zusammen Lysepuffer). Ernten Sie die Zellen mit einer Zelle scrapper und schnell übertragen Hälfte des Lysats auf jeweils zwei 1,5 ml Mikrozentrifugenröhrchen auf Eis. </li><li> Ein Branson Sonifier 150 wird verwendet, um die Zellwände aufbrechen. Legen Sie die Mikrozentrifugenröhrchen mit dem Zelllysat auf Eis und Impuls für 5 Sek., 5 Sek. und 5 Sek. mit Ultraschall-Einstellungen von 2, 2 bzw. 4. </li><li> Centrifuge das Zelllysat bei 12.500 RPM für zwei 4 Minuten dreht sich bei 4 ° C. Nach dem ersten Spin, vorsichtig umdrehen den Rohren, die lose Trümmer von der Seitenwand des Rohres zu entfernen. Nach dem zweiten Spin Transfer vorsichtig den Überstand ab, ohne dass das Pellet aus den beiden Röhren ein neues Mikrozentrifugenröhrchen auf Eis. </li><li> Berechnen Sie die Licht-Einheiten (GVE) je pl Lysat. Zur Messung der LU, verdünnte 1 ul Lysat mit 8 ul PBS in einem neuen Mikrozentrifugenröhrchen. Direkt mit 100 ul 1X Coelenterazin Substrat, um die verdünnte Mischung und sofort messen Lumineszenz in der Röhre mit einem Rohr Luminometer (20/20 n Turner Scientific) mit einem 5 Sekunden gelesen. </li><li> Bewahren Sie die Ruc-Antigen-Lysat bei -20 ° C für 1-2 Tage oder für einen längeren Zeitraum, wie oft in Aliquots bei -80 ° C. </li></ol> TEIL 3: Vorbereiten eines Sera Masterplatte <ol><li> Machen Sie eine sera Master Platte hergestellt, indem zunächst 450 ul Puffer A (50 mM Tris, pH 7,5, 100 mM NaCl, 5 mM MgCl 2, 1% Triton X-100) in jede Vertiefung einer 96-Deep-Well Polypropylen Mikrotiterplatte . Bei diesem Schritt kann ein Farbstoff, Phenolrot, auch in Puffer A aufgenommen werden, (Endkonzentration liegt bei 0,2 pg / ml in Puffer A) als Tracer für die Überwachung künftiger sera Probenzugabe und andere Schritte der LIPS-Assay zu handeln, um. </li><li> Als nächstes fügen Sie 50 ul Serum von jeder Probe, die verschiedenen Vertiefungen mit 450 ul Puffer A. Hinweis: Dies ist eine 1:10 Verdünnung der Seren in Puffer A. In der Regel Seren wird nicht auf die letzten beiden Vertiefungen der Master-Platte gegeben, weil Dies ist für den Puffer Rohlinge vorbehalten. </li><li> Vor der Verwendung der Master-Platte, ist es ausgiebig geschüttelt (1-2 Stunden) auf einem Rotator-Plattform. Das Serum in die Master-Platte ist staBLE für mindestens 1 Monat (oder länger) bei 4 ° C, wenn sie richtig, um Verdunstung zu verhindern gespeichert. </li><li> Wie unten beschrieben, bietet das Master-Platte 10 ul der verdünnten Seren wiederholt für die Profilierung der Seren gegen mehrere Antigene entfernt werden. Größere und kleinere Master-Platten können ebenfalls eingesetzt werden. Verschließen Sie die Platte gut mit Parafilm, wenn es nicht benutzt wird. </li></ol> TEIL 4: LIPS-Test <ol><li> Polypropylen 96-flachen Mikrotiterplatten werden verwendet, um sera Test. Im ersten Schritt, dann werden 40 ul Puffer A zu jedem Well der 96-Well-Platte mit einem 8-Kanal Mikropipette. </li><li> Nehmen Sie als nächstes 10 ul der verdünnten Seren (1 ul sera gleichwertig) von der Master-Platte und fügen Sie ihn direkt in jede Vertiefung der Arbeitsplatte mit einem 8-Kanal Mikropipette. </li><li> Ein Master-Mix mit den Ruc-Antigen-Extrakt ist neben solchen, die 1 x 10 7 light units (LU) in 50 ul Puffer A in jede Vertiefung gegeben formuliert. Lower-Eingänge (nur 1 X 10 6) können ebenfalls verwendet werden, sondern führen zu einer geringeren dynamischen Bereichs. Machen Sie dieses Master-Gemisch und Pipette 50 ul Ruc-Antigen-Gemisch in jede Vertiefung. </li><li> Die Platte auf einem Schüttler für 1 Stunde bei Raumtemperatur. </li><li> Während der Inkubation mit 5 ul einer 30% igen Suspension von Ultralink Protein A / G-Beads (Pierce Biotechnology, Rockford, IL) in PBS auf den Boden jeder Vertiefung einer neuen 96-Loch-Filter HTS-Platte (Millipore, Bedford, MA) . </li><li> Nach dem 1 Stunde Inkubation, übertragen Sie die 100 ul Ruc-Antigen-Antikörper-Reaktion Mischung auf 96-Well-Filter HTS-Platten, die das Protein A / G Kügelchen mit einem 8-Kanal Mikropipette. </li><li> Inkubieren Sie die 96-Well-Filterplatte auf einem Schüttler für 1 weitere Stunde bei Raumtemperatur. </li><li> Next waschen Sie den Filter Platte auf einem Vakuumkammer. Jedes der gut ist 8-mal mit 100 ul Puffer A gewaschen, gefolgt von zwei Mal mit 100 ul PBS. Dies kann manuell oder mit einem Roboter Pipettieren Workstation durchgeführt werden. </li><li> Nach der letzten Waschung wird das Vakuum abgeschaltet. Entfernen Sie die Filterplatte und blot es trocken mit einem Stapel von Papiertüchern oder Filterpapier achten Sie darauf, Feuchtigkeit auf der Ober-und Unterseite der Platte zu entfernen. </li></ol> TEIL 5: Messung der Lumineszenz und Datenanalyse Set-up: A Berthold LB 960 Centro Mikroplatten-Luminometer ist für die Bestimmung Lumineszenz in jede Vertiefung mit einer einzigen Düse verwendet. Nachdem die Maschine eingeschaltet ist, spülen Sie den Injektor mit destilliertem H 2 O mit dem Injektor Waschgang. Coelenterazin Substrat hergestellt unter Verwendung des Promega Renilla Substrat-Kit, wie vom Hersteller beschrieben. In der Regel 6 ml 1X Coelenterazin Substrat-Mix (dh 60 ul Coelenterazin Lager plus 6 ml 1X Puffer) ist für die Grundierung der Maschine vorbereitet und läuft eine volle 96-Well-Platte. Vor einer Analyse der Platte ist der Berthold LB 960 Centro Mikroplatten-Luminometer mit 1X Coelenterazin Substrat grundiert. Öffnen Sie ein Programm-Datei mit den Einstellungen für die Injektion des Substrates und das Lesen der Platte. Für diese Messungen wurden 50 ul Coelenterazin Substrat gespritzt wird, ist die Platte für 2 sec, durch ein 5 sec von Lumineszenz lesen gefolgt erschüttert. <ol><li> Obwohl die Platte Luminometer hat die Fähigkeit, die gesamte Platte zu lesen, kann eine partielle Lese von der Platte auch gewählt werden (unter der Lese-Menü) werden. Starten Sie das Programm, das von der Platte lesen initiiert. </li><li> Nach dem Lauf entfernen Sie die Mikrotiterplatte Filterplatte umgehend Verschütten in das Luminometer zu verhindern. Exportieren Sie die Daten mit dem MikroWin Programm in eine Excel-Format für die Analyse generiert. </li><li> Wir empfehlen die Beurteilung der Seren von zwei unabhängigen läuft. Die Daten werden dann gemittelt, um die LU-Titer-Werte für jede Probe zu erzeugen. Diese Werte können weiter angepasst, um den Puffer Rohlinge subtrahieren werden. </li><li> Zusätzliche Datenanalyse wie die Bestimmung der Cut-off aus dem Mittelwert plus 3 oder 5 Standardabweichungen der Steuerung Werte berechnet werden können. </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 erfordert nur minimale Assay-Optimierung und wegen seiner Einfachheit, qualitativ hochwertige Daten können in der Regel unter Verwendung LIPS in weniger als zwei Wochen für ein bestimmtes Antigen. Die zeitaufwendige Schritte sind das Klonen und die Erzeugung der entsprechenden Plasmid-Expressionsvektor mit der RUC-Antigen-Fusion. Sobald diese Plasmide erzeugt werden, können einzelne oder mehrere Antigene rasch mit LIPS getestet werden, wie oben beschrieben. Anzumerken ist, dass LIPS sehr vielseitig ist, dass Seren können auch in einem Rohr-Format oder alternativ auf einem Mikrotiter-Filterplatte mit Hilfe eines BIOMEK Roboter-Arbeitsstation 2 oder einem Teller Scheibe mit Vakuumfiltration getestet werden. Diese hoch skalierbare System ermöglicht die parallele LIPS Profilierung von einer Gruppe von Menschen Autoantigene 3 oder das gesamte Proteom eines Virus 4. Es ist wahrscheinlich, dass viele verschiedene Antikörper-Profile von LIPS generiert wird nützlich sein, für die Diagnostik 3-9, Entdeckung von Antigenen 9, nach dem Verlauf einer Behandlung 10, Impfstoff Überwachung und hat weitreichende Auswirkungen für bestimmte Erkrankungen und für die öffentliche Gesundheit im Allgemeinen.

<!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)

Technologien für umfassend zu verstehen und zu quantifizieren Antikörper-Profile auf Autoantigene und Infektionserreger können neue Einblicke in die Krankheitsmechanismen Ertrag und können neue Marker für Krankheiten aufzuklären mit unterschiedlichen klinischen Merkmalen substratify und besser zu verstehen Pathogenese. Wir haben eine sehr quantitative Methode aufgerufen Luciferase Immunpräzipitation Systems (LIPS) zur Profilierung Patientenseren Antikörper-Antwort auf Autoantigene und Erreger-Antigenen mit einer Infektion assoziiert entwickelt. Im Gegensatz zu ELISAs, ist das hoch empfindliche Lippen leicht implementiert, um humoralen serologischen Antwort Profile verschiedener Antigene in einem universellen Format Umfrage und produziert dynamische Antikörper-Titer im Bereich von bis zu 6 log<sub&gt; 10</sub&gt; Für einige Antigene. In diesen Studien wird die quantitative Profilierung von LIPS von Patienten humorale Reaktionen gegen Platten von Antigenen oder sogar das gesamte Proteom von einigen Krankheitserregern (zB HIV), in der Regel aussagekräftiger als Tests ein einziges Antigen mittels ELISA. Darüber hinaus LIPS eliminiert auch Zeit und den Aufwand zu hoch gereinigten Antigene sowie die arbeits-Assay Optimierungsschritte für Standard-ELISAs benötigt werden. Hier bieten wir Ihnen ein detailliertes Protokoll beschreibt die technischen Aspekte der Durchführung LIPS-Assays für leicht Profilierung Antikörper-Antworten auf einzelne oder mehrere Antigene.

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

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

Die technischen Aspekte der Durchführung LIPS (Luciferase Immunpräzipitation Systems) beschrieben sind. Das Gesamtkonzept beinhaltet Ausdruck chimären Gene Antigene fusioniert an Renilla Luziferase (RUC) in Säugerzellen. Crude Ruc-Antigen-Extrakte werden dann aufbereitet und ohne Reinigung eingesetzt in Immunpräzipitationsassays, um Antikörper zu quantifizieren.

Antikörper Profiling von Luciferase Immunpräzipitation Systems (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 Aufrufe.  National Institute of Dental and Craniofacial Research, National Institutes of Health. Die technischen Aspekte der Durchführung LIPS (Luciferase Immunpräzipitation Systems) beschrieben sind. Das Gesamtkonzept beinhaltet Ausdruck chimären Gene Antigene fusioniert an Renilla Luziferase (RUC) in Säugerzellen. Crude Ruc-Antigen-Extrakte werden dann aufbereitet und ohne Reinigung eingesetzt in Immunpräzipitationsassays, um Antikörper zu quantifizieren.

Serie: 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.

AC elektrokinetischen Phänomenen von Mikroelektroden-Strukturen erzeugt

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, ...

Forschung

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).

Methylierte DNA Immunpräzipitation

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: Immunpräzipitation mittels Durchflusszytometrie Detected

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.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

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.

Chemisch-Antikörper blockiert Microarray für Multiplex-High-Throughput-Profiling von spezifischen Protein-Glykosylierung bei 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 ...

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.

Induktion und Analyse von epithelialen zu mesenchymalen 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 der Translation Initiation Während Stress-Bedingungen durch Polysomprofile 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 Individuelle humanen embryonalen Stammzellen durch quantitative 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.

Bewertung der Selective mRNA Translation in Säugerzellen durch Polysomen Profilieren

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 Glucocorticoid-Analyse: Nicht-invasive Monitoring-Adrenal in Equids

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