Wir danken Servane Contal und Boris Untereiner für ihre technischen Beitrag leisten. Wir erkennen die Unterstützung der FNR &quot;FUTOX&quot;-Projekt.

Das Protokoll benötigt 16 Tage für seine Vollendung. Der Zeitrahmen ist in Abbildung 1 dargestellt sind. Details zum Puffer Vorbereitung <ul><li> Vorbereitung der Lyse-Puffer (3 ml pro Probe). </li><li> Vorbereitung der Waschpuffer (50 mL pro Probe). Dieser Puffer sollte vorgekühlt werden und bei -20 ° C gelagert in der Tiefkühltruhe bis zur weiteren Analyse und auf Eis gehalten während des gesamten Verfahrens. </li><li> Vorbereitung des Niederschlags-Puffer (20 ml pro Probe). Dieser Puffer sollte vorgekühlt werden und bei -20 ° C gelagert in der Tiefkühltruhe, bis es gebraucht und auf Eis gehalten während des gesamten Verfahrens. </li><li> Die Arbeiten sollten vorzugsweise in die kalte Kammer durchgeführt werden bei 4 ° C. </li></ul> Pilzarten Drei Stämme für Proteom-Analysen verwendet wurden klassifiziert, um Fusarium graminearum auf der Grundlage ihrer morphologischen Merkmale und Translation-Elongationsfaktor Alpha-1-Analyse (O&#39;Donnell et al., 1998) Diese Stämme gehörten zu den CRP-Gabriel Lippmann Sammlung und wurden gepflegt und gelagert bei - 80 ° C in 15% Glycerin. Vorbereitung der Myzelien Pilze wurden auf V8 für 4 Tage bei 25 ° C abwechselnd helle und dunkle Periode jeweils 12 Stunden kultiviert. Die Kulturen wurden fragmentiert mit einem sterilen Messer und Stücke von diesen Kulturen wurden verwendet, um 250 ml Erlenmeyerkolben mit 100 ml Toxin-induzierenden Medien zu impfen. Myzelien wurden nach 10 Tagen geerntet und von Medium durch Filtrieren der Kultur mit einer sterilen Filter abgeschieden. Myzelien wurden 3 mal mit sterilem Wasser gewaschen, um mittel-Rückständen und anderen Verbindungen zu entfernen. Wir haben dann sofort die Zellen durch Zugabe von flüssigem Stickstoff eingefroren und hielt die Proben bei -80 ° C. Allgemeine Beschreibung der Protein-Extraktions- Protein-Proben wurden extrahiert nach der Methode in der Figur 3A und 3B beschrieben. Der Fusarium-Proben wurden in flüssigem Stickstoff gemahlen und das Pulver wurde in 10 ml Teflon-Röhrchen gesammelt. Die Proben wurden dann 30 Minuten mit dem Lysepuffer inkubiert, 10 Minuten gekocht und zentrifugiert, 2 mal bei Raumtemperatur (12000g für 15 Minuten). Die Überstände wurden gesammelt und bei -20 ° C inkubiert mit Niederschlag Puffer über Nacht. Nach Zentrifugation bei 4 ° C 30000g für 45 Minuten, die Pellets der gefällten Proteine ​​wurden 3 mal mit kaltem Aceton enthält DTT gewaschen. Schließlich wurden die Pellets an der Luft getrocknet und die Proteine ​​wurden bei der Kennzeichnung Puffer resolubilisiert, Einstellung des pH auf 8. Eine Teilprobe von 30 ml wurde für Gesamt-Protein Quantifizierung entfernt und der verbleibende Überstand wurde bei -20 ° C bis Protein-Elektrophorese gespeichert. <img src="/files/ftp_upload/1690/1690fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Timeline des Verfahrens. <img src="/files/ftp_upload/1690/1690fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Scheme für mehrere Betreiber Probe-Verarbeitung. <img src="/files/ftp_upload/1690/1690fig3atmb.jpg" alt="Abbildung 3a" border="0" /> Abbildung 3a. <img src="/files/ftp_upload/1690/1690fig3btmb.jpg" alt="Abbildung 3b" border="0" /> Abbildung 3b. Bitte <a href="/files/ftp_upload/1690/1690fig3a.jpg">klicken Sie hier,</a> um eine größere Version der Figur 3a, oder sehen <a href="/files/ftp_upload/1690/1690fig3b.jpg">Sie hier</a> für eine größere Version Bild 3b. Abb. 3a-b Flussdiagramme für die Protein-Extraktions-Verfahren (A: am ersten Tag; B: zweiter Tag).. <img src="/files/ftp_upload/1690/1690fig4tmb.jpg" alt="Abbildung 4" border="0" /> Abbildung 4. 1D Bild von Protein-Extrakten. Die Proteine ​​wurden bis zur vollständigen Migration von blau bis zum Ende des Gels, die mit Lava Lila gefärbten ausgeführt wurde. Bitte <a href="/files/ftp_upload/1690/1690fig4.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 4 zu sehen.

<ol>
	<li>Bridge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Extraction+from+Fungi.">Protein Extraction from Fungi.</a> Protein Purification Protocols. , 39-48 (1996).</li><li>Jiao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+carbon+sources+on+trichothecene+production+and+Tri+gene+expression+by+Fusarium+graminearum+in+liquid+culture.">Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture.</a> FEMS Microbiol Lett. 285, 212-219 (2008).</li><li>Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+filamentous+fungi.">Proteomics of filamentous fungi.</a> Trends in Biotechnology. 25, 395-400 (2007).</li><li>Milles, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+proteomic+approach+to+monitor+protein+synthesis+in+mycotoxin+producing+moulds.">Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds.</a> Mycotoxin Res. 23, 161-165 (2007).</li><li>Morrison, N., Field, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concept+of+sample+in+OMICS+technology.">Concept of sample in OMICS technology.</a> OMICS. 10, 127-137 (2006).</li><li>O'Donnell, K., Kistler, H. C., Cigelnik, E., Ploetz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+evolutionary+origins+of+the+fungus+causing+Panama+disease+of+banana:+Concordant+evidence+from+nuclear+and+mitochondrial+gene+genealogies.">Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies.</a> PNAS. 95, 2044-2049 (1998).</li><li>Paper, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomics+of+extracellular+proteins+in+vitro+and+in+planta+from+the+pathogenic+fungus+Fusarium+graminearum.">Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum.</a> Proteomics. 7, 3171-3183 (2007).</li><li>Pasquali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video+in+science.+Protocol+videos:+the+implications+for+research+and+society.">Video in science. Protocol videos: the implications for research and society.</a> EMBO Rep. 8, 712-716 (2007).</li><li>Taylor, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimum+information+about+a+proteomics+experiment+(MIAPE).">The minimum information about a proteomics experiment (MIAPE).</a> Nat Biotechnol. 25, 887-893 (2007).</li><li>Taylor, 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=Proteomic+analyses+of+Fusarium+graminearum+grown+under+mycotoxin-inducing+conditions.">Proteomic analyses of Fusarium graminearum grown under mycotoxin-inducing conditions.</a> Proteomics. 8, 2256-2265 (2008).</li></ol>

Das gegenwärtige Interesse an Proteomik Ansätze innerhalb der Pilz-Biologie-Domain zu einer erhöhten Zahl von Veröffentlichungen mit dieser Technik (wie in Kim et al., 2007 reviewed) geführt. Verfahren zur Extraktion der Proteine ​​setzen auf eine Kombination von Extraktionsverfahren, sie sind oft kaum in der methodischen Abschnitten beschrieben und erfordern Fachkenntnisse im Umgang mit den potentiellen Methoden zur Fehlerbehebung. Wie bei anderen &quot;omics&quot; Ansätze, Standards für die Probe und Datenverarbeitung ist, um verlässliche wissenschaftliche Daten zu generieren unerlässlich. Außerdem Proben und Verfahren der Manipulation soll adäquat beschrieben werden, um gemeinsame Interpretation der Ergebnisse zwischen den verschiedenen &quot;omics&quot; Experimente erlauben. Dies wäre wichtig für eine optimale Nutzung Potentialität der Cross-Data-Mining in der Genomik, Proteomik, Metabolomik, ... (Morrison et al., 2006). Minimum Informationen über ein Proteom-Experiment wurde ausgearbeitet und Arbeitsgruppen wurden eingerichtet, um alle Aspekte eines Experiments (Gel-Elektrophorese, Massenspektrometrie, Molekulare Wechselwirkungen, Protein Modifikationen, Proteomics Informatik, Sample Processing) zu bewältigen. Zur Zeit sind keine definierten Informationen finden Sie auf Probe Verarbeitungsverfahren. Angesichts der Bedeutung der Protein-Extraktions-Verfahren bei der Bestimmung der endgültigen Qualität der Proteom-Studien, um die Verfahren, dass die Standardisierung im Rahmen der MIAPE Richtlinien zu erhöhen (Taylor et al., 2007) implementieren können, haben wir vorgeschlagen, die Verwendung eines Video-Protokoll detailliert Probe Verarbeitung. Video Beschreibung von Experimenten kann wesentlich dazu beitragen, die Zahl der Informationen über die Probenaufbereitung, die in einer besseren Reproduzierbarkeit der &quot;omics&quot; Experimente (Pasquali, 2007) Ergebnis erhöhen können. In der Tat ist die Reproduzierbarkeit in Labors eine Grundvoraussetzung, um die Gültigkeit Ergebnisse aus der Proteomik (http://www.fixingproteomics.org) zu gewährleisten. Verschiedenen Besetzungen und verschiedenen Betreibern Manipulation Proben: Das Kreuz-Labor-Reproduzierbarkeit wird durch zwei Faktoren beeinflusst. In dem beschriebenen Protokoll, schlagen wir vor, führen biologische Replikate mit mehreren Betreibern (Abbildung 2 und Video), um die Zuverlässigkeit der Daten (dh technische Variation der Ergebnisse ist zu berücksichtigen) zu erhöhen. Das Verfahren zur Extraktion der Proteine ​​beschrieben wird, auf SDS und Heizung auf. Dies war bisher gezeigt, dass eine gute Reinheit und Menge der Proteine ​​(Bridge 1996) zu gewährleisten. SDS mit kochendem gekoppelt löst die Zellwände, hydrophobe Proteine ​​und verhindert die Bildung von Oligomeren, die abort Fällung von Proteinen. Boiling ermöglicht auch die Inaktivierung von Proteasen. Der gleiche Effekt wird auch durch EDTA, PMSF und komplette Mini-Protease-Inhibitor produziert. DTT entfernt Disulfidbrücken in und zwischen Proteinen erleichtern Protein Solubilisierung. Um SDS vor IEF zu entfernen, sind Proteine, die durch Aceton / TCA / DTT gefällt. Darüber hinaus ermöglicht Aceton / TCA / DTT Niederschlag Entfernen einiger Schadstoffe wie Lipide, Nukleinsäuren, Salze und / oder Phenol-Verbindungen, wenn diese Verbindungen vorhanden sind. Wie SDS, verhindern, dass diese Moleküle eine gute Migration während IEF. Nach diesem Niederschlag, ist es notwendig, die ausgefallenen Proteine ​​durch Aceton / DTT, zu waschen, um TCA zu entfernen, da es mit IEF stören können. Eine gute Vorbereitung der Proben ist der Schlüssel zu guten Ergebnissen. Dafür ist es auch wichtig, um eine Verunreinigung von Proteinen aus dem Umfeld der Arbeit mit puderfrei Handschuhe und Respekt für eine gute Laborpraxis zu vermeiden. Aus technischer Sicht innerhalb der beschriebenen Protokoll werden die wichtigsten Schritte für den Erhalt ausreichender Menge und Reinheit von Proteinen in zwei Phasen. Erstens, die Schleif-Phase, in der völligen Zerstörung der Zelle ist von grundlegender Bedeutung, um Proteine ​​Release, und zweitens, die Reinigung des gesamten Proteine, eine Phase umfasst die Entfernung von der Mehrheit der Lipide, DNA und andere Verunreinigungen, die mit der Migration der Proteine ​​stören können.

Medien und Lösungen PDA: 39 g Kartoffel-Dextrose-Agar (Difco), 1 L destilliertem Wasser. V8-Agar: 200 ml V8 (Campbell, USA), 2g CaCO3 (Sigma), 16g Agar (Difco), 800 ml destilliertem Wasser Toxin induziert Medien: 1g K 2 HPO 4, 0,5 g KCl, 0,5 g MgSO 4 7H 2 O, 10 mg FeEDTA, 2g L Glutaminsäure, 10g Saccharose in 1L destilliertes Wasser. Lysis Buffer: <table border="1"><tr><td></td><td> 100ml </td><td> Endkonzentration </td><td></td></tr><tr><td> Tris-HCL pH 8 </td><td> 5 ml </td><td> 50mM </td><td> 1M </td></tr><tr><td> SDS </td><td> 2g bzw. 10 mL </td><td> 2% </td><td> 20% </td></tr><tr><td> DTT </td><td> 500ml </td><td> 10 mM </td><td> 2M </td></tr><tr><td> EDTA </td><td> 20mL </td><td> 0,1 mM </td><td> 0,5 M </td></tr><tr><td> PMSF </td><td> 200ml </td><td></td><td></td></tr><tr><td> Komplett mini Protease-Inhibitor </td><td> 1 Tablette </td><td></td><td></td></tr><tr><td> Wasser </td><td> Qsp </td><td></td><td></td></tr></table> Niederschlag Buffer: <table border="1"><tr><td></td><td> 100ml </td><td> Endkonzentration </td></tr><tr><td> TCA </td><td> 20mL </td><td> 20% </td></tr><tr><td> DTT </td><td> 0,1 ml </td><td> 0,1% </td></tr><tr><td> Aceton </td><td> Qsp </td><td></td></tr></table> Waschpuffer: <table border="1"><tr><td></td><td> 100ml </td><td> Endkonzentration </td></tr><tr><td> DTT </td><td> 0,1 ml </td><td> 0,1% </td></tr><tr><td> Aceton </td><td> 100ml </td><td></td></tr></table> Kennzeichnung Buffer (Lagerung bei -18 ° C in kleinen Portionen): <table border="1"><tr><td></td><td> 1ml </td><td> Endkonzentration </td></tr><tr><td> Harnstoff </td><td> 140 mg </td><td> 7M </td></tr><tr><td> Thioharnstoff </td><td> 50 mg </td><td> 2M </td></tr><tr><td> CHAPS </td><td> 40 mg </td><td> 4% </td></tr><tr><td> Tris </td><td> 30 ul </td><td> 30 mM </td></tr><tr><td> Wasser </td><td> 1 mL </td><td></td></tr></table>

toxin induction and protein extraction from fusarium spp. cultures for proteomic studies

Fusarien sind Fadenpilze in der Lage, verschiedene Toxine produzieren. Fusarium Mykotoxinen wie Deoxynivalenol, Nivalenol, T2, zearelenone, fusaric Säure, Moniliformin, etc. .. nachteilige Auswirkungen auf Mensch und Tier Gesundheit und einige werden als Pathogenitätsfaktoren angesehen. Proteomik-Studien erwies sich als wirksam zur Entschlüsselung Toxinproduktion Mechanismen (Taylor et al., 2008) sowie zur Identifizierung potentieller pathogenen Faktoren (Paper et al., 2007, Houterman et al., 2007) in Fusarien. Es wird daher von grundlegender Bedeutung für zuverlässige Methoden für den Vergleich zwischen Proteom-Studien, um auf wahren Unterschiede in der Proteinexpression zwischen den Experimenten, Zerrungen und Labors gefunden verlassen zu etablieren. Das Verfahren, das beschrieben werden soll, um ein höheres Maß an Standardisierung der Proteom-Verfahren auf zwei Arten beitragen. Die gefilmten Protokoll wird verwendet, um das Niveau der Details, die genau beschrieben werden können, erhöht. Darüber hinaus bildet die Verfügbarkeit von standardisierten Verfahren zur Verarbeitung biologischer sollte eine höhere Robustheit der Daten zu gewährleisten, auch unter Berücksichtigung des menschlichen Faktors im Rahmen der technischen Reproduzierbarkeit der Extraktion. Das beschriebene Protokoll benötigt 16 Tage für seine Fertigstellung: 14 Tage für Kulturen und zwei Tage für die Protein-Extraktion (Abbildung 1). Kurz gesagt, Fusarium-Stämmen auf festen Medien für 4 Tage gewachsen, sie sind dann manuell fragmentiert und in eine modifizierte Toxin induziert Medien (Jiao et al, 2008.) Für 10 Tage. Myzel durch Filtration durch ein Miracloth Schicht gesammelt. Schleifen wird in einer Kältekammer durchgeführt. Verschiedene Betreiber durchgeführt Extraktion Wiederholungen (n = 3), um unter Berücksichtigung der Voreingenommenheit aufgrund technischer Veränderungen (Abbildung 2). Die Extraktion wurde auf einem SDS / DTT-Puffer wie in Taylor et al beschrieben ist. (2008) mit leichten Modifikationen. Insgesamt Proteinextraktion erforderte eine Fällung der Proteine ​​mit Aceton / TCA / DTT-Puffer über Nacht und Aceton / DTT Waschen (Abbildung 3a, 3b). Die Proteine ​​wurden schließlich in der Protein-Kennzeichnung Puffer resolubilisiert und quantifiziert. Die Ergebnisse der Extraktion wurden visualisiert auf einem 1D-Gel (Abbildung 4, SDS-PAGE), bevor Sie mit 2D-Gelen (IEF / SDS-PAGE). Das gleiche Verfahren kann für Proteom-Analysen auf anderen Kultursubstrate und andere Fadenpilze (Miles et al., 2007) angewendet werden.

Watch this Scientific Journal Video about Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies at JoVE.com

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein-Extraktion für Proteom-Analysen in Pilzarten erfordert ein hohes Maß an Standardisierung zu erfolgen gemäß den Mindestanforderungen Informationen über ein Proteom-Experiment (MIAPE) Richtlinien. Wir präsentieren ein Video-Protokoll, das ein Verfahren zur Minimierung der experimentellen Bias während Toxin Induktion und Extraktion der Proteine ​​aus schließt<em&gt; Fusarium spp.</em

Toxin Induktions-und Protein-Extraktion aus Fusarium Spp. Kulturen für Proteom-Studien

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

toxin-induction-protein-extraction-from-fusarium-spp-cultures-for

Nanyang Technological University

Research

JoVE Journal

Biology

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

16.9K Aufrufe.  Centre de Recherche Public-Gabriel Lippmann. Protein-Extraktion für Proteom-Analysen in Pilzarten erfordert ein hohes Maß an Standardisierung zu erfolgen gemäß den Mindestanforderungen Informationen über ein Proteom-Experiment (MIAPE) Richtlinien. Wir präsentieren ein Video-Protokoll, das ein Verfahren zur Minimierung der experimentellen Bias während Toxin Induktion und Extraktion der Proteine ​​aus schließt Fusarium spp.

Serie: Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-Dissektion Rattenhirns RNA oder Protein Extraction von bestimmten Hirnregion

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Forschung

Biologie

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Elektrophoretische Trennung von Proteinen

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

Interview: Proteinfaltung und Studien für Neurodegenerative Erkrankungen

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

Herstellung von Ratten-Gehirn-Aggregate Kulturen für Neuron und Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Digitale Mikrofluidik für die automatisierte Verarbeitung Proteomic

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Mit dem GELFREE 8100 Fraktionierung System für Molecular Weight-Based Fraktionierung mit Liquid Phase Wiederherstellung

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

DNA-Extraktion aus in Paraffin eingebettete Material für genetische und epigenetische Analysen

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

Sexuelle Entwicklung und Ascosporenausschleuderung in Fusarium graminearum</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naive Adult Stem Cells Isolation von primären humanen Fibroblasten Kulturen

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

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 ...