Wir sind dafür, dass wir, um die Fluoreszenz-Spektrometer zu nutzen und für seine Hilfe danken der Dr. Thierry Pourcher. Das Labor wird von der Ligue Nationale Contre le Cancer (équipe einem Gütezeichen) gefördert, der Verband pour la Recherche contre le Cancer, das Institut National du Cancer (INCa), der Agence Nationale pour la Recherche, METOXIA (EU FP7-Programm), das Zentrum A. Lacassagne, das Centre National de la Recherche Scientifique, das Institut National de la Santé et de la Recherche Médicale und der Universität Nizza ( <a href="http://www.unice.fr/isdbc/" target="_blank">http://www.unice.fr/isdbc/</a> ). Wir danken Dr. Christiane Brahimi M-Horn für das kritische Lesen und redaktionelle Korrektur.

Biochemische Titration von Glykogen 1. Zelllyse <ol><li> Samenzellen in einer Konzentration von 0,5-2 x 10 6 pro 100 mm Schale. </li><li> Behandlung: inkubieren Zellen 24, 48 oder 72 Stunden in niedriger Sauerstoffkonzentration (Hypoxie) in einem Bug-Box anaerobe Arbeitsstation (Ruskinn Technologie Biotrace International Plc, Bridgend, UK) bei 1% oder 0,1% O 2, 94% oder eingestellt 94,9% N &amp;sub2; und 5% CO 2. Parallel inkubieren Zellen in Normoxie (21% O 2, 5% CO 2). Mediumwechsel (25 mM Glucose-haltigem Medium) alle 24 h auf Schwankungen in der Glukosekonzentration während der Zeit des Experiments zu minimieren. </li><li> Nach der Behandlung, Wasch Zellen 2x mit PBS, um Spuren von Glukose aus dem Kulturmedium zu entfernen. Kratzen Sie die Zellen in kaltem PBS. </li><li> Zentrifugieren Sie die Lösung bei 1000 rpm für 5 min, den Überstand verwerfen und waschen das Pellet einmal mit PBS. Das Pellet bei -20 ° C eingefroren, bevor weiter fortgefahren werden.</li><li> Resuspendieren des Pellets in 100 ul destilliertem Wasser. 100 l des Glykogen-Hydrolyse-Puffer mit der Glykogen-Assay Kit (Abcam) vorgesehen ist. Man kocht das Lysat bei 95 ° C für 5 min, um Enzyme zu inaktivieren. </li><li> Vortex-Zentrifuge und das Lysat bei 13.000 rpm für 10 min, um die unlöslichen Produkte zu entfernen. Halten Sie den Überstand. </li><li> Glykogen Ebenen auf den Proteingehalt zwischen den Proben zu normalisieren, kann die Quantifizierung von Proteinen mit 25-35 ul des Überstands unter Verwendung des Bicinchoninsäure-Assays (BCA-Interchim) erfolgen. </li></ol> Einige der Lysat kann verwendet werden, um die freie Glucose-Konzentration in den Zellen zu messen. 2. Hydrolyse von Glykogen <ol><li> Mischungs 50 ul des Überstands (Schritt 1) ​​mit 1 &amp;mgr; l der Hydrolyse Enzyme Mix. Dieses Gemisch enthält Glucoamylase. Inkubation für 30 min bei Raumtemperatur. </li><li> Bereiten Sie das Kalibrierungskurve durch Verdünnen der Standard Glykogen (2 mg / ml) smit dem Kit zu einer Lösung von 10 ug / ml in dem Puffer upplied Hydrolyse. Als nächstes bereiten sechs getrennten Röhrchen mit 0, 4, 8, 12, 16 und 20 ul von 10 ug / ml-Standard und justieren Sie jedes Röhrchen bis zu einem Endvolumen von 50 ul mit Hydrolyse-Puffer auf eine Konzentration von Glykogen von 0 zu geben, 0,04 , 0,08, 0,12, 0,16 und 0,2 ug / ml. 1 ul Enzym-Hydrolyse-Mischung in den Standards und Inkubation für 30 min bei Raumtemperatur. </li></ol> Der Hintergrund Glukosekonzentration der Zelllinie, nach Wert für die freie Glucose-Konzentration gegeben werden, aus dem Wert für die Gesamtglucosekonzentration (Glukose aus dem Glykogen Hydrolyse + free-Glucose), um das Niveau von Glykogen in der Zelle bestimmen, subtrahiert werden. 3. Entwicklung und Lesen von Fluoreszenz <ol><li> Für jeden Zustand, bereiten ein Rohr (Rohr 1) in freie Glucose-Konzentration und zwei Röhren zu messen (Rohre 2 und 3) Gesamtglucosekonzentration (jedes Rohr Witz messenha verschiedenen Volumen von hydrolysierten Glykogen). </li><li> Hinzufügen von 15 &amp;mgr; l Überstand am Ende von Schritt 1 in Rohr 1 extrahiert. Hinzufügen von 35 &amp;mgr; l Puffer, um die Hydrolyse bis zu einem Endvolumen von 50 ul zu vervollständigen. Erhalten Fluoreszenz gibt die freie Glucose-Konzentration. </li><li> In X ul hydrolysierten Glykogen (in Schritt 2) erhalten, auf Rohr 2. Einstellen, um ein Endvolumen von 50 ul. Probenvolumen (X ul) ist entsprechend der Zelldichte und / oder Zelltyp, um Fluoreszenzwerte, die nicht über die Konzentrationen der Kalibrierungskurve zu erhalten eingestellt werden. </li><li> In 2 X ul hydrolysierten Glykogen in Röhre 3. Einstellen, um ein Endvolumen von 50 ul. </li><li> Bereiten Sie eine Entwicklungslösung für Proben und Standards, indem man für jedes Rohr 48,7 ul Entwicklung Buffer, 1 ul der Entwicklung Enzyme Mix und 0,3 ul OXIRED Probe (in der Glykogen-Assay Kit geliefert). Zum Beispiel für zwei Bedingungen, bereiten Sie einen Mix für 12 Rohrs (6-Standards, 2 für freie Glucose und 4 für die Gesamtglucose Fluoreszenz). </li><li> Dann werden 50 ul dieser Mischung zu jedem Röhrchen und Inkubation für 30 min bei Raumtemperatur im Dunkeln. </li><li> Messung der Fluoreszenz bei einer Anregungswellenlänge von 535 nm, einer Emissionswellenlänge von 587 nm, wie empfohlen, und einen Schlitz von 3 nm für die Anregung und Emission. </li></ol>

<ol>
	<li>Pelletier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+Synthesis+is+Induced+in+Hypoxia+by+the+Hypoxia-Inducible+Factor+and+Promotes+Cancer+Cell+Survival.">Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival.</a> Front Oncol. 2, (2012).</li><li>Alonso, M. D., Lomako, J., Lomako, W. M., Whelan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+the+biogenesis+of+glycogen.">A new look at the biogenesis of glycogen.</a> FASEB J. 9, 1126-1137 (1995).</li><li>Bollen, M., Keppens, S., Stalmans, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+features+of+glycogen+metabolism+in+the+liver.">Specific features of glycogen metabolism in the liver.</a> Biochem. J. 336 (Pt 1), 19-31 (1998).</li><li>Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J., Stapleton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP-activated+protein+kinase+does+not+associate+with+glycogen+alpha-particles+from+rat+liver.">AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver.</a> Biochem. Biophys. Res. Commun. 362, 811-815 (2007).</li><li>Sullivan, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+alpha+and+beta+particles+in+glycogen+using+molecular+size+distributions.">Nature of alpha and beta particles in glycogen using molecular size distributions.</a> Biomacromolecules. 11, 1094-1100 (2010).</li><li>Chee, N. P., Geddes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+liver+glycogen.">The structure of liver glycogen.</a> FEBS Lett. 73, 164-166 (1977).</li><li>Roach, P. J., Depaoli-Roach, A. A., Hurley, T. D., Tagliabracci, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+and+its+metabolism:+some+new+developments+and+old+themes.">Glycogen and its metabolism: some new developments and old themes.</a> Biochem. J. 441, 763-787 (2012).</li><li>Moses, S. W., Bashan, N., Gutman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+metabolism+in+the+normal+red+blood+cell.">Glycogen metabolism in the normal red blood cell.</a> Blood. 40, 836-843 (1972).</li><li>Agbanyo, M., Taylor, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+3-deoxy-3-fluoro-D-glucose+into+glycogen+and+trehalose+in+fat+body+and+flight+muscle+in+Locusta+migratoria.">Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle in Locusta migratoria.</a> Biosci. Rep. 6, 309-316 (1986).</li><li>Louzao, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Fluorescent+glycogen"+formation+with+sensibility+for+in+vivo+and+in+vitro+detection.">"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection.</a> Glycoconj. J. 25, 503-510 (2008).</li><li>Sheehan, D. C. H., B, 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> Theory and practice of histotechnology. , (1980).</li><li>Baba, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+monoclonal+antibody+that+recognizes+glycogen+and+its+application+for+immunohistochemistry.">Production of monoclonal antibody that recognizes glycogen and its application for immunohistochemistry.</a> Kokubyo Gakkai Zasshi. 60, 264-287 (1993).</li></ol>

Die Autoren erklären, keinen Interessenkonflikt.

Biochemische Titration von Glykogen in vitro ermöglicht eine genaue Quantifizierung von Zellen Glykogen. Im Vergleich zu einigen anderen Techniken (PAS, Immunfluoreszenz mit einem Glykogen-Antikörper, usw..) Ist diese Titration sehr spezifisch, empfindlich und reproduzierbar. Darüber hinaus ist das Verfahren bequemer, da es keine Radioaktivität aber eine Fluoreszenz-Spektrometer erfordert. Jedoch ist diese Technik rein quantitativ und liefert keine Informationen über Glykogen Verteilung in der Zel...

Glycogen ist ein mehrfach verzweigten Polymer von Glucose-Resten, die im Zytoplasma von vielen Zelltypen vorhanden ist. Es ist eine der Hauptformen der Energiespeicher in Zellen und spielt eine wichtige Rolle in den Glukosestoffwechsel. Die meisten Säugetierzellen sind in der Lage, und speichern Glykogen, die schnell in Glukose abgebaut können Glykolyse und ATP-Produktion während der metabolischen Stress fördern zu produzieren. Hepatozyten produzieren riesige Mengen von Glykogen, um den Blutzuckerspiegel, wodurch eine kontinuierliche Zufuhr von Glucose, um den Körper zu regulieren. Im Gegensatz dazu ist die Konzentration von Glykogen in anderen Zellen (Muskeln, rote Blutkörperchen, etc.) relativ gering. Jedoch lokal ausreichend sind, um Energie in der kurzfristigen, wenn die Zellen einmal in eine Umgebung von Nährstoffen entzogen ausgesetzt sind diese Mengen. Glykogen-Synthese und Glykogen-Abbau folgt den gleichen Schritten in allen Geweben (Abbildung 1). Erstens Glucosetritt Zellen durch Glucose-Transporter (GLUTs), und wird schnell von der Glucose-6-Phosphat (G6P) in Glucose-1-phosphat (G1P) durch Phosphoglucomutase umgewandelt. G1P wird dann in UDP-Glucose geändert, und das C-C1 von UDP-Glucose auf einen Tyrosinrest glycogenin, dem Verankerungsprotein von Glykogen befestigt. Dieses Molekül, als ein Primer Glykogen wird durch die Befestigung des UDP-Glucose an die terminale Glucose durch eine α (1 → 4)-Bindung über die Glykogensynthase verlängert. Schließlich wird, wenn eine lineare Kette von mehr als 11 Glucoseresten gebildet wird, das Verzweigungsenzym trägt ein Endgerät Oligosaccharid von einem Minimum von 6 Glucosereste an einer anderen Zuckerkettensuche durch eine α (1 → 6)-Bindung gebildet wird. Die Wiederholung dieses Prozesses gibt eine massive fraktalen Struktur, die Zweige, die eine Helix mit 6,5 Glukose pro Runde bilden. Glykogen kann umgekehrt hydrolysiert werden, um durch die konzertierte Aktion Glucosevon entzweigenden Enzymen, die α (1 → 6) verbunden und Glykogen Phosphorylase, die die α (1 → 4)-glycosidisch gebundene zwischen dem letzten Rest von Glucose aus einem Zweig und dem Glykogenmoleküls hydrolysiert hydrolysieren. Diese Reaktion nennt Glycogenolyse durch die Erhöhung von AMP (reflektierende ATP-Verbrauch) aktiviert und durch Glukose und ATP 2,3 gehemmt. Durch Elektronenmikroskopie wurden Glykogen Moleküle in vielen Zelltypen als freie Teilchen β (oder Glykogen Monopartikel) von 15-30 nm im Durchmesser beschrieben. In bestimmten Zelltypen, wie Hepatozyten, können β-Teilchen in eine komplexe zusammengebaut werden, um Rosetten, auch bekannt als α-Teilchen, die im Durchmesser variieren von 80 nm zu einem Maximum von 200 nm 4 bilden </sup&gt;. Die Art, wie diese β-Teilchen gebunden sind, größere Cluster von α-Teilchen zu bilden, ist noch nicht vollständig aufgeklärt. Einige Beweise scheint zu beweisen, daß β-Teilchen können durch kovalente Bindung 5, Wasserstoffbrücken oder auch durch Protein-Protein-Wechselwirkungen 6 verklebt werden. Die Menge von Glykogen in den Zellen gespeichert ist abhängig von vielen Parametern: (I) die Menge des glycogenin in der Zelle, die Glykogensynthese initiiert, (ii) die Aktivität von Glykogen-Synthase-Phosphorylase und durch Protein-Phosphorylierung / Dephosphorylierung reguliert, (iii) die Konzentration Glucose in den Zellen, die abhängig von verschiedenen Parametern wie beispielsweise die Bereitstellung von Glucose aus dem Gefäßsystem und die Glucoseaufnahme durch die Zellen ist. Glykogenspeicher sind eng durch allosterische Regulation der Biosynthese Hormone über Zwischenmetabolite reguliert durch Hormone regulieren den Energiestoffwechsel und durch Nährstoffsensorsignalwege 7. Es ist wichtig,Lage, Glykogen in biologischen Proben zu quantifizieren, um die Bedeutung der Glykogen-Stoffwechsel im ganzen Körper und zellulärer Ebene zu verstehen. Wir beschreiben hier eine präzise, ​​reproduzierbare und bequem biochemischen in vitro-Assay für Glykogen. Diese Technik beruht auf der Quantifizierung Glucose Fluoreszenz vor und nach der spezifischen Hydrolyse von Glykogen basiert. Es gibt andere Methoden, um das Niveau von Glykogen in den Zellen zu schätzen, aber die meisten von ihnen sind nicht quantitativ. Eine der ersten für die Quantifizierung von Glykogen in den Zellen beschriebenen Verfahren wurde der Messung der [14 C]-Glucose in Glykogen Einbau 8,9 basiert. Die Verwendung von Radioaktivität macht dieses Verfahren schwieriger zu handhaben, sondern es den Vorteil, dass die Rate der Glukose in Glykogen Einbau und bei der Unterscheidung zwischen der Verteilung der Glucosereste an den äußeren Ästen und in dem Kern des Moleküls (es erfordert auch eine zusätzliche β-amylolysis und einem chromatographischen Schritt). Eine andere Technik wurde in jüngerer Zeit entwickelt und beruht auf dem Einbau von 2-NBDG basiert (2 - {N-[7-Nitrobenz-2-oxa-1 ,3-diazolon-4-yl] amino}-2-Desoxyglucose), ein 2-Desoxyglucose fluoreszierendes Derivat, in Glykogen 10. Die gemessene Fluoreszenzintensität spiegelt die Menge an Glykogen hergestellt und kann mit einem Fluoreszenzlesegerät gemessen werden. Fluoreszenzverteilung in der Zelle kann durch konfokale Mikroskopie ausgewertet. Unter den anderen nicht-quantitative Techniken ist Periodic Acid-Schiff-Färbung (PAS) vielleicht die häufigste. Es kann für den Nachweis von Glykogen in fixierten Zellen, Gewebeschnitte in Paraffin oder gefroren werden. Diese histologischen Technik Farben nicht speziell Polysaccharide, Glykolipide, Glykoproteine, Zellulose und neutrale Muzine in Lila. Die Spezifität des Tests kann durch Behandlung der fixierten Zellen oder Gewebeschnitten mit Diastase, die spezifisch verdaut Glykogen erhöht werden. Danachdas Niveau von Glykogen kann qualitativ durch den Vergleich nicht hydrolysierten Proben (alle Kohlenhydrat modifizierter Makromoleküle) zu hydrolysierten Proben (Kohlenhydrat-modifizierten Makromolekülen außer Glykogen) geschätzt werden. PAS-Färbung und mikroskopische Analyse, im Gegensatz zu biochemischen Assays von Glykogen, enthält Informationen über die Verteilung von Glykogen in der Zelle, die diffundiert oder in einem bestimmten Teil der Zelle konzentriert werden können. Obwohl PAS-Färbung Schätzungen Unterschiede in Glykogen Akkumulation zwischen verschiedenen Bedingungen ist es jedoch nicht quantitativ 11. Ein monoklonaler Maus-Antikörper, ursprünglich unter Verwendung Kiefer Kondylenknorpels als Antigen wurde gezeigt, dass mit Glykogen in den Zellen und mit gereinigtem Glykogen in vitro 12 reagieren. Da diese Antikörper spezifisch Glykogen-verwandte Zuckerketten, ist es ein nützliches Werkzeug für den Nachweis von Glykogen durch Immunhistochemie in einer spezifischeren Weise als die PAS-Färbung. </p&gt; Elektronenmikroskopie ist eine weitere Technik, die Visualisierung von Körnern von Glykogen in den Zellen und die Bewertung des Grades der Glykogen-Speicher ermöglicht. In der Tat sind Glykogen β-Teilchen leicht mit einem Elektronenmikroskop als elektronendichtes Granulat 1 erkennbar.

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			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>31966.047</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glycogen Assay Kit</td>
			<td>Abcam</td>
			<td>ab65620 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		<tr>
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 <td></td>
 <td>EQUIPMENT</td>
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			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
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		 <tr>
			<td>Fluorescence Spectrometer</td>
			<td>PerkinElmer</td>
			<td>LS 50B</td>
			<td></td>
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 </table>

biochemical titration of glycogen in vitro

Glycogen ist das Haupt energetische Polymer von Glucose in Wirbeltieren und spielt eine entscheidende Rolle bei der Ganzkörper-Stoffwechsels als auch im Zellstoffwechsel. Viele Verfahren zur Glykogen erkennen existieren bereits, aber nur wenige sind quantitativ. Wir beschreiben hier eine Methode mit dem Abcam Glykogen-Assay-Kits, die auf spezifischen Abbau von Glykogen basiert, um durch Glucoamylase Glucose. Glukose wird dann speziell auf ein Produkt, das mit dem OXIRED Sonde an Fluoreszenz zu erzeugen reagiert oxidiert. Die Titration ist präzise, ​​empfindlich und kann auf Zellextrakten oder Gewebeschnitten erreicht werden. Im Gegensatz zu anderen Techniken, es nicht geben Informationen über die Verteilung von Glykogen in der Zelle. Als ein Beispiel für diese Technik, beschreiben wir hier die Titration von Glykogen in beiden Zelllinien des Chinesischen Hamsters CCL39 Lungenfibroblasten und menschlichen Kolonkarzinom LS174, in Normoxie inkubiert (21% O 2) in Abhängigkeit von Hypoxie (1% O 2). Wir vermuten, dass Hypoxie ist eine signal, dass bereitet Zellen, um zu überleben 1 zu synthetisieren und zu speichern Glykogen.

Ein niedriger Gehalt an Sauerstoff (Hypoxie) in Tumoren Signale an Tumorzellen die Notwendigkeit, Energie zu speichern, um nachfolgende Nährstoffverarmung verarbeiten, um zu überleben. Glycogen ist das Haupt energetische Polymer von Glucose in Säugerzellen, untersuchten wir die Regulierung der Glykogen-Speicherung im Hypoxie. Die Berechnung und die Standardisierung der Konzentration von Glykogen in Zelllysaten müssen auf den Rohdaten der Fluoreszenz durchgeführt werden, wie in den Tabellen 1, 2 und...

Watch this Scientific Journal Video about Biochemical Titration of Glycogen In vitro at JoVE.com

Biochemical Titration of Glycogen In vitro

Wir beschreiben hier eine genaue, reproduzierbare und bequem biochemische Methode zur Titration von Glykogen in vitro. Diese Technik nutzt die Abcam Glykogen-Assay-Kit und ist auf aufeinander folgende Hydrolyse von Glykogen durch Fluoreszenz Glukose und Glukose-Titration.

Biochemische Titration von Glykogen<em&gt; In-vitro-</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

biochemical-titration-of-glycogen-in-vitro

Research

JoVE Journal

Biology

Biochemical Titration of Glycogen In vitro

28.3K Aufrufe.  University of Nice - Sophia Antipolis.  Wir beschreiben hier eine genaue, reproduzierbare und bequem biochemische Methode zur Titration von Glykogen in vitro. Diese Technik nutzt die Abcam Glykogen-Assay-Kit und ist auf aufeinander folgende Hydrolyse von Glykogen durch Fluoreszenz Glukose und Glukose-Titration. 

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Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

Titration von Human Coronaviren Mit einem Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

Forschung

Biologie

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafisch Gehirn Ventrikel Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

Sammeln Variable-Konzentration Isotherme Titrationskalorimetrie Datasets in Auftrag, um bindende Mechanismen bestimmen

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Biochemische Rekonstitution der Steroid-Rezeptor • Hsp90-Protein-Komplexen und Reaktivierung der Ligandenbindung

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Blutentnahme für Biochemische Untersuchungen an adulten Zebrafisch

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Biochemische und High Throughput Mikroskopische Beurteilung der Fettmasse in<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Biochemischen Assays zur Analyse von Aktivitäten von ATP-abhängigen Chromatin-Remodeling-Enzyme

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Hochdurchsatz-Titration von Luciferase-exprimierenden rekombinanten Viren

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Biochemische Titration von Glykogen

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