Wir sind dankbar, dass Matthew Robert Weatherhead für einen exzellenten technischen Service und John Ralph, University of Wisconsin für wertvolle Ratschläge, Diskussionen und die Pappel Probe. Diese Arbeit wurde vom US Department of Energy (DOE) Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494) und von der Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science finanziert , US Department of Energy (Preis-Nr. DE-FG02-91ER20021).

1. Zellwand Isolation <ol><li> Grind etwa 60-70mg von Luft-oder gefriergetrocknete Pflanzenmaterial mit 5,5 mm Edelstahl-Kugeln in einem 2 ml Sarstedt Schraubverschluss Röhrchen mit einem iWall, ein Mahlen und Abfüllen Roboter (30 s). Eine Alternative nicht-Roboter-low-Durchsatz-Verfahren unter Anwendung einer Kugelmühle (retschmill) ist in Teil II 2 dargestellt. </li><li> Fügen Sie 1,5 ml 70% wässrigem Ethanol auf die verzichtet Mahlgut und gründlich vortexen. </li><li> Zentrifuge bei 10.000 rpm für 10 min zur Pelletierung der Alkohol unlöslichen Rückstand. </li><li> Verwerfen Sie den Überstand. </li><li> Fügen Sie 1,5 ml Chloroform / Methanol (1:1 v / v) Lösung des Rückstandes und schütteln Rohr gründlich, um das Pellet zu resuspendieren. </li><li> Zentrifuge bei 10.000 rpm für 10 min und Verwerfen des Überstandes. </li><li> Pellet in 500 ul Aceton. </li><li> Das Lösungsmittel wird mit einem Luftstrom bei 35 ° C, bis es trocken. Bei Bedarf getrockneten Proben können bei Raumtemperatur bis zur weiteren Verarbeitung gespeichert werden. </li><li> Zur Einleitung der Entfernung von Stärke aus der Probe das Pellet in 1,5 ml einer 0,1 M Natriumacetat-Puffer pH 5,0. </li><li> Cap der Sarstedtröhrchen und Wärme für 20 min. bei 80 ° C in einem Heizblock. </li><li> Kühlen Sie die Suspension auf Eis. </li><li> Fügen Sie die folgenden Substanzen, um das Pellet: 35 ul von 0,01% Natriumazid (NaN 3), 35 ul Amylase (50 ug / mL H 2 O, von Bacillus-Arten, Sigma), 17 ul Pullulanase (17,8 Einheiten aus Bacillus acidopullulyticus; Sigma) . Cap der Röhre und gründlich vortexen. </li><li> Die Suspension wird über Nacht bei 37 ° C in den Shaker. Die Ausrichtung der Rohre horizontal Helfer eine bessere Durchmischung. </li><li> Hitze Suspension bei 100 ° C für 10 min in einem Heizblock, um die Verdauung zu kündigen. </li><li> Zentrifuge (10.000 rpm, 10 min) und Überstand verwerfen, die solubilisierten Stärke. </li><li> Waschen Sie die verbleibende Pellet dreimal durch Zugabe von 1,5 ml Wasser, Vortexen, Zentrifugieren und Dekantieren des Waschwassers. </li><li> Pellet in 500 ul Aceton. </li><li> Das Lösungsmittel wird mit einem Luftstrom bei 35 ° C, bis es trocken. Es kann notwendig sein auch zu brechen das Material in der Röhre mit einem Spatel für eine bessere Trocknung. Das getrocknete Material präsentiert isoliert Zellwand (Lignocellulose). Bei Bedarf getrockneten Proben können bei Raumtemperatur bis zur weiteren Verarbeitung gespeichert werden. </li></ol> 2. Ligningehalt Diese Methode basiert auf einem gemeldeten Methode Fukushima und Hatfield 3. <ol><li> Wiegen Sie 1 bis 1,5 mg vorbereitet Zellwandmaterial (siehe 1) in 2 ml-Messkolben der Ausfahrt aus einer Tube leer für einen Rohling. </li><li> Spülen Rohrwandungen mit 250 ul Aceton an die Zellwand Material auf den Boden des Röhrchens zu sammeln und verdampft das Aceton sehr sanft unter Luftstrom. </li><li> Vorsichtig mit 100 ul frisch gemacht Acetylbromid-Lösung (25% v / v Acetylbromid in Eisessig) entlang der Rohrwände um Spritzer zu vermeiden. </li><li> Cap-Messkolben und Wärme bei 50 ° C für 2 Stunden </li><li> Wärme für eine zusätzliche Stunde mit Vortexen alle 15 Minuten. </li><li> Cool auf Eis auf Raumtemperatur. </li><li> Add 400 ul 2M Natronlauge und 70 ul frisch zubereitete 0,5 M Hydroxylaminhydrochlorid. Vortex-Messkolben. </li><li> Füllen Sie Messkolben genau auf die 2,0 ml Marke mit Eisessig, verschließen und mehrmals zu mischen. </li><li> Je 200 ul der Lösung in eine UV spezifischen 96-Well-Platte und las in einem ELISA-Reader bei 280nm. </li><li> Bestimmen Sie den Prozentsatz der Acetylbromid Lignin (% ABSL) unter Verwendung eines geeigneten Koeffizienten (Pappel = 18.21; Grasses = 17,75; Arabidopsis = 15,69) mit folgender Formel: % ABSL Calc: <img src="/files/ftp_upload/1745/1745eq.jpg" alt="Abbildung 1" /> Multiplikation% ABSL mit 10 Treffern in der ug / mg Zellwand-Einheit Es hilft zu tun mindestens 3 Platte liest die Absorption (abs), da Partikel können eine leichte Variation in Extinktionswerte Ursache Durchschnitt. Hinweis: 0,539 cm stellt die Schichtdicke, sondern in Abhängigkeit von der Platte könnte dies müssen bestimmt werden. </li></ol> 3. Ligninzusammensetzung Diese Methode ist aus einer neueren Methode von Robinson und Mansfield 5 veröffentlicht hat. <ol><li> Etwa 2 mg der Zellwand-Material (siehe 1.) In einer Schraube verschlossen Glasrohr für thioacidolysis. </li><li> sorgfältig vorbereiten 2,5% Bortrifluoriddiethyletherat (BF 3), 10% Ethanthiol (EtSH)-Lösung. Sie müssen einen Luftballon mit Stickstoff gefüllt, um die verlorenen Volumens im Dioxan Flasche mit Stickstoff zu verdrängen. Dioxan ist sehr gefährlich, nicht nehmen Proben oder Geräte aus der Haube. 20 pl EtSH;; 5 pl BF 3 175 ul Dioxan: Volumes für die Vorbereitung der Lösung pro Probe benötigt. </li><li> Add 200 ul EtSH, BF 3, Dioxanlösung zu jeder Probe. </li><li> Purge Vial Kopfraum mit Stickstoffgas und sofort wieder aufsetzen. </li><li> Hitze auf 100 ° C für 4 Stunden unter leichtem Mischen jede Stunde. </li><li> End Reaktion durch Kühlen auf Eis für 5 Minuten. </li><li> Add 150 ul 0,4 M Natriumbicarbonat, Wirbel </li><li> Für den Clean-up 1 ml Wasser und 0,5 ml Ethylacetat, Wirbel und lassen Phasen zu trennen (Ethylacetat auf, Wasser auf der Unterseite). </li><li> Transfer 150 ul des Ethylacetatschicht in ein 2 ml Sarstedt Röhrchen. Achten Sie darauf, kein Wasser übertragen wird. </li><li> Dampft Lösungsmittel durch einen Konzentrator mit Luft. </li><li> Geben Sie 200 ul Aceton und verdampft (Wiederholung für insgesamt zwei Mal zu entfernen überschüssiges Wasser). </li><li> Für die TMS-Derivatisierung add 500 ul Ethylacetat, 20 ul Pyridin, und 100 ul N, O-Bis (trimethylsilyl) acetamid in jedes Röhrchen. </li><li> inkubieren für 2 Stunden bei 25 ° C. </li><li> Transfer-100 ul der Reaktion in ein GC / MS Fläschchen und 100 ul Aceton. </li><li> Analysieren Sie die Proben durch GC mit einem Quadrupol-Massenspektrometer oder Flammenionisationsdetektor ausgestattet. Ein Agilent HP-5ms-Säule installiert ist (30 mm x 0,25 mm X 0,25 um Filmdicke). Die folgenden Temperaturgefälle ist mit einem 30 min Lösungsmittel Verzögerung und einem 1,1 ml / min Durchfluss verwendet: Initial halten bei 130 ° C für 3 min; 3 ° C / min Rampe zu einer 250 ° C und für 1 min halten; ermöglichen Equilibrierung auf die ursprüngliche Temperatur von 130 ° C. </li><li> Peaks werden von relativen Retentionszeiten mit Tetracosan interner Standard (optional) oder durch charakteristische Massenspektrum Ionen von 299 identifizierten m / z, 269 m / z und 239 m / z für S, G, H und Monomere sind (siehe Abb.. 2). Die Zusammensetzung des Lignin-Komponenten wird durch die Einstellung der gesamten Peakfläche um 100% quantifiziert </li></ol> 4. Repräsentative Ergebnisse Ein Beispiel für eine Wand-Analyse ist in Abbildung 2 dargestellt. In diesem Fall Pappel Stamm (Holz) wurde durch die verschiedenen Verfahren in das Protokoll Abschnitt beschrieben analysiert. Ein Beispiel-Chromatogramm der Trennung von Lignin-Komponenten nach thioacidolysis und TMS-Derivatisierung wird angezeigt. Offensichtlich ist die relative Häufigkeit von Syringyl-(S), Guaiacyl-(G) und p-hydroxyphenol-(H)-Einheiten bestimmt werden kann. Für den Inhalt der Acetylbromid Lignin ist selbsterklärend, kann man erwarten, Werte zwischen 20-50% der Wand Trockengewicht. Man sollte beachten, dass Acetylbromid nicht lösen alle das Lignin in der Wand, und dass der Grad der Solubilisierung kann variieren je nach Material. Allerdings ist dieses Verfahren relativ einfach durchzuführen und eine schnelle und gibt einen hervorragenden Angleichung der Ligningehalt in einer Lignocellulose-Material. <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="Abbildung 1" /> Abbildung 1:. Übersicht von Lignocellulose-Analyse Zellwände (Lignocellulose) werden aus Rohöl getrocknetes Pflanzenmaterial isoliert. Die Wand wird anschließend in Aliquots gewichtet und unterteilt für die verschiedenen Tests. Das Wandmaterial ist mit Acetylbromid und das gelöste Lignin durch UV-Spektroskopie quantifiziert behandelt. Für die Bestimmung der Ligninzusammensetzung ist Wandmaterial thioacidolysis unterzogen. Die solubilisierten Phenole unterziehen TMS-Derivatisierung und kann dann getrennt und quantifiziert werden durch GC-MS-Analyse. Die Matrix Polysaccharid Zusammensetzung und kristalline Cellulose-Gehalt Protokoll wird in Teil II 2 diskutiert. <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="Abbildung 2" /> Abbildung 2:. Umfassende lignocellulosischen Analyse von Pappelholz Hackschnitzel aus Pappel (Populus tremoloides) wurden zu den beschriebenen Protokollen unterzogen. Ligin Zusammensetzung; H p-hydroxyphenyl; G Guaiacyl; S Syringyleinheiten.

<ol>
	<li>Carroll, A., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulosic+Biofuels.">Cellulosic Biofuels.</a> Annual Review of Plant Biology. 60, 165-165 (2009).</li><li>Foster, C. E., Martin, T., Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+compositional+analysis+of+Plant+Cell+Walls+(Lignocellulosic+biomass),+Part+II:+Carbohydrates.">Comprehensive compositional analysis of Plant Cell Walls (Lignocellulosic biomass), Part II: Carbohydrates.</a> J Vis Exp. , (2010).</li><li>Fukushima, R. S., Hatfield, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+isolation+of+lignin+for+utilization+as+a+standard+to+determine+lignin+concentration+using+the+acetyl+bromide+spectrophotometric+method.">Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method.</a> J. Agric. Food Chem. 49 (7), 3133-3133 (2001).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+carbohydrates+and+their+modification+as+a+resource+for+biofuels.">Cell-wall carbohydrates and their modification as a resource for biofuels.</a> Plant J. 54 (4), 559-559 (2008).</li><li>Robinson, A. R., Mansfield, S. 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+analysis+of+poplar+lignin+monomer+composition+by+a+streamlined+thioacidolysis+procedure+and+near-infrared+reflectance-based+prediction+modeling.">Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling.</a> Plant J. 58 (4), 706-706 (2009).</li><li>Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+systems+approach+to+understanding+plant-cell+walls.">Toward a systems approach to understanding plant-cell walls.</a> Science. 306 (5705), 2206-2206 (2004).</li><li>Teeri, T. T., Brumer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+characterization+and+applications+of+enzymes+from+the+wood-forming+tissues+of+poplar:+Glycosyl+transferases+and+xyloglucan+endo-transglycosylases.">Discovery, characterization and applications of enzymes from the wood-forming tissues of poplar: Glycosyl transferases and xyloglucan endo-transglycosylases.</a> Biocatalysis and Biotransformation. 21, 173-173 (2003).</li></ol>

Die beschriebenen Methoden ermöglichen eine schnelle quantitative Beurteilung der Ligningehalt und Zusammensetzung von Lignocellulose-Biomasse-Anlage. Mit dem iWall Roboter ca. 350 Proben können Boden und abgegeben werden pro Tag. Der Durchsatz der verschiedenen analytischen Methoden pro Person variiert. Mit der beschriebenen Protokolle hier aus können 30 Proben für Ligningehalt verarbeitet werden, und 15 für Ligninzusammensetzung pro Tag. Durch die quantitative Natur der Daten optimal Rohstoff Getreide können Sorte oder Genotypen in Bezug auf ihre Eignung für die Produktion von Biokraftstoffen bewertet werden.

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<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>Hydroxylamine Hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>255580</td>
<td>&nbsp;</td>
<tr>
<td>Acetyl Bromide</td>
<td>&nbsp;</td>
<td>Aldrich</td>
<td>135968</td>
<td>&nbsp;</td>
<tr>
<td>Ethanethiol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E3708</td>
<td>&nbsp;</td>
<tr>
<td>Borontrifluoride diethyl etherate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>15719</td>
<td>&nbsp;</td>
<tr>
<td>N,O,-Bis(trimethylsilyl) acetimide</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>15241</td>
<td>&nbsp;</td>
<tr>
<td>Dioxane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>296309</td>
<td>&nbsp;</td>
<tr>
<td>Spectromax Plus 384</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Plus384</td>
<td>&nbsp;</td>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>6890 GC/5975B MSD</td>
<td>(lignin composition)</td>
</tr>
<tr>
<td>5.5mm Stainless Steel Balls</td>
<td>&nbsp;</td>
<td>Salem Ball Company</td>
<td>(N/A)</td>
<td>&nbsp;</td>
<tr>
<td>96 well plate heat spreader</td>
<td>&nbsp;</td>
<td>Biocision</td>
<td>Coolsink 96F</td>
<td>&nbsp;</td>
<tr>
<td>Heating block</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>Dri-block DB-3D</td>
<td>&nbsp;</td>
<tr>
<td>Sample concentrator</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>FSC400D</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part i: lignin

Der Bedarf an erneuerbaren, klimaneutralen und nachwachsenden Rohstoffen für die Industrie und die Gesellschaft hat sich zu einem der drängendsten Probleme des 21. Jahrhunderts. Dies hat das Interesse an der Verwendung von pflanzlichen Erzeugnissen als industrielle Rohstoffe für die Produktion von flüssigen Kraftstoffen für den Transport neu entfacht<sup&gt; 1</sup&gt; Und andere Produkte wie Biokomposit Materialien<sup&gt; 7</sup&gt;. Pflanzlicher Biomasse bleibt eines der größten unerschlossenen Reserven auf dem Planeten<sup&gt; 4</sup&gt;. Es ist vor allem der Zellwände, die von energiereichen Polymeren, einschließlich Cellulose, Hemicellulosen verschiedenen (Matrix Polysaccharide und das Polyphenol Lignin sind zusammen<sup&gt; 6</sup&gt; Und damit manchmal Lignocellulose bezeichnet. Allerdings haben pflanzlichen Zellwände entwickelt, um widerspenstig, um den Abbau als Wände Zugfestigkeit bereitzustellen, um Zellen und die gesamte Pflanzen, Pathogene abzuwehren, und damit das Wasser in der Pflanze transportiert werden; im Fall von Bäumen bis zu mehr die 100 m über Erdgeschoss. Aufgrund der vielfältigen Funktionen von Wänden, gibt es eine immense strukturelle Vielfalt innerhalb der Mauern der verschiedenen Pflanzenarten und Zelltypen innerhalb einer einzigen Anlage<sup&gt; 4</sup&gt;. Daher, je von dem, was Pflanzenarten und Abwechslung, oder Pflanzengewebe ist für eine Bioraffinerie verwendet wird, müssen die Verarbeitungsschritte für die Depolymerisation durch chemische / enzymatische Prozesse und die anschließende Vergärung der verschiedenen Zuckerarten auf flüssige Biokraftstoffe angepasst und optimiert werden. Diese Tatsache unterstreicht die Notwendigkeit, für eine gründliche Charakterisierung pflanzlicher Biomassen. Hier beschreiben wir eine umfassende analytische Methode, die Bestimmung der Zusammensetzung von Lignocellulose ermöglicht und ist offen für eine mittel-bis High-Throughput-Analyse. In diesem ersten Teil haben wir auf die Analyse der Polyphenol Lignin (Abbildung 1) zu konzentrieren. Das Verfahren beginnt mit der Vorbereitung der Stärke befreite Zellwandmaterial. Die daraus resultierende Lignocellulose werden dann bis zu seinem Ligningehalt von acetylbromide Solubilisierung bestimmen split<sup&gt; 3</sup&gt;, Und seine Ligninzusammensetzung in Bezug auf seine Syringyl, Guaiacyl-und p-hydroxyphenyl Einheiten<sup&gt; 5</sup&gt;. Das Protokoll für die Analyse der Kohlenhydrate in Lignozellulose einschließlich Cellulose-Gehalt und Matrix Polysaccharidzusammensetzung ist in Teil II diskutiert<sup&gt; 2</sup&gt;.

Watch this Scientific Journal Video about Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin at JoVE.com

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Pflanzlicher Biomasse ist ein wichtiger klimaneutrale erneuerbare Ressource, die für die Herstellung von Biokraftstoffen verwendet werden könnten. Pflanzliche Biomasse besteht im Wesentlichen aus Zellwände, eine strukturell komplexen Verbundwerkstoff bezeichnet Lignocellulose. Hier beschreiben wir ein Protokoll für eine umfassende Analyse des Inhalts und der Zusammensetzung der Polyphenol-Lignin.

Umfassende Analyse der Zusammensetzung von pflanzlichen Zellwänden (Lignozellulose) Teil I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

comprehensive-compositional-analysis-plant-cell-walls-lignocellulosic

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JoVE Journal

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Cell Biology

Biology

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

Unit 1

Plant Cell Structure and Organization

SCIENTISTS IN ACTION

32.0K Aufrufe.  Michigan State University (MSU). Pflanzlicher Biomasse ist ein wichtiger klimaneutrale erneuerbare Ressource, die für die Herstellung von Biokraftstoffen verwendet werden könnten. Pflanzliche Biomasse besteht im Wesentlichen aus Zellwände, eine strukturell komplexen Verbundwerkstoff bezeichnet Lignocellulose. Hier beschreiben wir ein Protokoll für eine umfassende Analyse des Inhalts und der Zusammensetzung der Polyphenol-Lignin.

Serie: Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Menschliche Inselzellen der Bauchspeicheldrüse Isolation: Part I: Verdauung und Sammlung von Pankreasgewebe

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

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Biologie

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 2

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 3

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

Umfassende Analyse der Zusammensetzung von pflanzlichen Zellwänden (Lignozellulose) Teil II: Kohlenhydrate

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

Label-freie In situ Imaging der Verholzung in pflanzlichen Zellwänden

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Glycan Profiling of Plant Cell Wall Polymers mit Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabolische Markierung und Membran Fraktionierung für Vergleichende Proteomanalyse von<em&gt; Arabidopsis thaliana</em&gt; Fahrwerk Zellkulturen

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Eine effiziente Methode zur quantitativen, Einzel-Zell-Analyse von Chromatin Modifikation und Reaktor Architektur in Whole-Mount-Samenanlagen in<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

Isolierung und Analyse der Zusammensetzung von Pflanzen Cuticle Lipid Polyester Monomere

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...