Ringraziamo Servane Contal e Boris Untereiner per il loro contributo tecnico. Riconosciamo l&#39;appoggio di FNR progetto &quot;FUTOX&quot;.

Il protocollo richiede 16 giorni per il suo completamento. Il calendario dettagliato è in figura 1. Dettagli per la preparazione del buffer <ul><li> Preparazione del tampone di lisi (3 ml per campione). </li><li> Preparazione del tampone di lavaggio (50 mL per campione). Questo buffer deve essere pre-refrigerati e conservati a -20 ° C nel freezer fino a quando ulteriori analisi e tenuta in ghiaccio durante l&#39;intera procedura. </li><li> Preparazione del tampone precipitazioni (20 mL per campione). Questo buffer deve essere pre-refrigerati e conservati a -20 ° C nel freezer fino al momento e tenuta in ghiaccio durante l&#39;intera procedura. </li><li> Il lavoro dovrebbe preferibilmente essere effettuata nella camera fredda a 4 ° C. </li></ul> Specie fungine Tre ceppi utilizzati per le analisi proteomiche sono state classificate da Fusarium graminearum sulla base delle loro caratteristiche morfologiche e Traduzione-Allungamento Alfa-1 fattore di analisi (O&#39;Donnell et al., 1998) Questi ceppi appartenevano alla CRP-Gabriel Lippmann e la raccolta sono stati mantenuti e conservati a - 80 ° C in 15% glicerolo. Preparazione di miceli Funghi sono stati coltivati ​​su V8 ​​per 4 giorni a 25 ° C si alternano periodo di chiari e scuri ogni 12 ore. Le culture sono state frammentate con una lama sterile e pezzi di queste culture sono state utilizzate per inoculare beute da 250 ml contenente 100 ml di tossine che inducono i media. Miceli sono state raccolte dopo 10 giorni e sono stati separati dai media filtrando la cultura con un filtro sterile. Miceli sono stati lavati 3 volte con acqua sterile per rimuovere i residui di medie e di altri composti. Abbiamo quindi immediatamente congelato le cellule con l&#39;aggiunta di azoto liquido e conservati i campioni a -80 ° C. Descrizione generale di estrazione di proteine Campioni di proteine ​​sono stati estratti con il metodo descritto nella figura 3A e 3B. I campioni sono stati Fusarium terreno in azoto liquido e la polvere sono stati raccolti in 10 ml tubi in Teflon. I campioni sono stati poi incubati 30 minuti con il tampone di lisi, bollita per 10 minuti, 2 volte e centrifugati a temperatura ambiente (12000g per 15 minuti). I surnatanti sono stati raccolti e incubate a -20 ° C con buffer di precipitazioni durante la notte. Dopo centrifugazione a 4 ° C 30000g per 45 minuti, il pellet di proteine ​​precipitate sono state lavate 3 volte con acetone freddo contenente DTT. Infine, i pellet sono stati essiccati e le proteine ​​erano resolubilized nel buffer di etichettatura, la regolazione del pH a 8. Un sub-campione di 30 ml è stato rimosso per la quantificazione delle proteine ​​totali e il surnatante rimanente è stato conservato a -20 ° C fino elettroforesi delle proteine. <img src="/files/ftp_upload/1690/1690fig1.jpg" alt="Figura 1" /> Figura 1. Temporale della procedura. <img src="/files/ftp_upload/1690/1690fig2.jpg" alt="Figura 2" /> Figura 2. Schema per operatori multipli campione di elaborazione. <img src="/files/ftp_upload/1690/1690fig3atmb.jpg" alt="Figura 3a" border="0" /> Figura 3a. <img src="/files/ftp_upload/1690/1690fig3btmb.jpg" alt="Figura 3b" border="0" /> Figura 3b. Si prega di <a href="/files/ftp_upload/1690/1690fig3a.jpg">cliccare qui</a> per vedere una versione più grande figura 3a, oppure <a href="/files/ftp_upload/1690/1690fig3b.jpg">qui</a> per una versione più grande di figura 3b. Figure 3a-b diagrammi di flusso per la procedura di estrazione delle proteine ​​(A: primo giorno; B: secondo giorno).. <img src="/files/ftp_upload/1690/1690fig4tmb.jpg" alt="Figura 4" border="0" /> Figura 4. 1D immagine di estratti proteici. Le proteine ​​sono stati eseguiti fino migrazione completa del blu fino alla fine del gel che era macchiata di viola lava. Si prega di <a href="/files/ftp_upload/1690/1690fig4.jpg">cliccare qui</a> per vedere una versione più grande della figura 4.

<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>

Recente interesse per approcci di proteomica all&#39;interno del dominio della biologia fungina ha portato ad un aumento del numero di pubblicazioni utilizzando questa tecnica (come recensione a Kim et al., 2007). Metodi per l&#39;estrazione delle proteine ​​si basano su una combinazione di procedure di estrazione, ma sono spesso scarsamente descritti nelle sezioni metodologiche e richiedono esperienza nel trattare con i metodi di risoluzione dei problemi potenziali. Come per gli altri &quot;omiche&quot; si avvicina, definizione di standard per l&#39;elaborazione dei campioni e dei dati è essenziale al fine di generare informazioni scientifiche affidabili. Inoltre i campioni e le procedure di manipolazione deve essere adeguatamente descritte in modo da consentire l&#39;interpretazione risultato condiviso tra diversi &quot;omiche&quot; esperimenti. Questo sarebbe essenziale per sfruttare a pieno le potenzialità di cross-data mining nel campo della genomica, proteomica, metabolomica, ... (Morrison et al., 2006). Informazioni minime su un esperimento di proteomica è stato redatto e gruppi di lavoro sono stati creati al fine di affrontare tutti gli aspetti di un esperimento (gel elettroforesi, spettrometria di massa, le interazioni molecolari, modifiche delle proteine, Informatica Proteomica, Sample Processing). Al momento non sono disponibili informazioni definite sulle procedure di trattamento del campione. Data l&#39;importanza delle procedure di estrazione delle proteine ​​nel determinare la qualità finale di studi di proteomica, al fine di attuare procedure che possono aumentare di normalizzazione nel quadro di linee guida MIAPE (Taylor et al., 2007), abbiamo proposto l&#39;uso di un protocollo video detailing sample elaborazione. Descrizione del video di esperimenti possono contribuire ad aumentare considerevolmente il numero di informazioni sul trattamento dei campioni che possono risultare in una migliore riproducibilità dei &quot;omiche&quot; esperimenti (Pasquali, 2007). Infatti, la riproducibilità attraverso laboratori è un requisito fondamentale per garantire la validità dei risultati proteomica (http://www.fixingproteomics.org). Il cross-laboratorio riproducibilità è influenzata da due fattori: strumentazioni differenti e differenti operatori manipolare campioni. Nel protocollo descritto, ci proponiamo di svolgere biologici replica che coinvolge più operatori (Figura 2 e video) di aumentare l&#39;affidabilità dei dati (cioè variazione tecnica dei risultati viene preso in considerazione). La procedura descritta per l&#39;estrazione di proteine ​​si basa sulla scheda di sicurezza e riscaldamento. Ciò è stato già dimostrato di garantire una buona purezza e la quantità di proteine ​​(Ponte 1996). SDS accoppiato con bollente si scioglie pareti delle cellule, delle proteine ​​idrofobiche e previene la formazione di oligomeri che le precipitazioni abortire delle proteine. Ebollizione consente anche l&#39;inattivazione delle proteasi. Lo stesso effetto viene prodotto anche con EDTA, PMSF e completa inibitore della proteasi mini. DTT rimuove ponti disolfuro tra le proteine ​​e facilitare la solubilizzazione delle proteine. Al fine di rimuovere SDS prima IEF, le proteine ​​vengono precipitate con l&#39;acetone / TCA / DTT. Inoltre, acetone / TCA / DTT precipitazioni permette la rimozione di alcuni contaminanti come i lipidi, acidi nucleici, i sali e / o composti fenolici, quando questi composti sono presenti. Come la SDS, queste molecole prevenire una migrazione bene durante IEF. A seguito di questa precipitazione, è necessario lavare le proteine ​​precipitate con l&#39;acetone / DTT, in modo da eliminare TCA in quanto può interferire con IEF. Una buona preparazione del campione è la chiave per un buon risultato. Per questo, è anche essenziale per evitare la contaminazione delle proteine ​​dall&#39;ambiente di lavoro con i guanti senza polvere e nel rispetto buone pratiche di laboratorio. Da un punto di vista tecnico, all&#39;interno del protocollo descritto, le fasi più critiche di ottenere quantità sufficienti e la purezza delle proteine ​​sono due fasi. In primo luogo, la fase di macinazione, dove completa distruzione delle cellule è fondamentale per liberare le proteine, e la seconda, la purificazione di proteine ​​totali, una fase che comprende la rimozione della maggior parte dei lipidi, DNA e altri contaminanti che potrebbero interferire con la migrazione delle proteine.

Mezzi di comunicazione e soluzioni PDA: 39 g di patate Destrosio Agar (Difco), 1 l di acqua distillata. V8 agar: 200 ml V8 (Campbell, USA), 2g CaCO3 (Sigma), Agar 16g (Difco), 800 ml di acqua distillata Tossine che inducono i media: 1g K 2 HPO 4, 0,5 g di KCl, 0,5 g MgSO 4 7H 2 O, 10 mg FeEDTA, 2g di L acido glutammico, saccarosio 10 g in 1 litro di acqua distillata. Lysis buffer: <table border="1"><tr><td></td><td> 100 ml </td><td> Concentrazione finale </td><td></td></tr><tr><td> Tris-HCL pH8 </td><td> 5ml </td><td> 50mM </td><td> 1M </td></tr><tr><td> SDS </td><td> 2g o 10 ml </td><td> 2% </td><td> 20% </td></tr><tr><td> DTT </td><td> 500mL </td><td> 10mM </td><td> 2M </td></tr><tr><td> EDTA </td><td> 20 mL </td><td> 0.1mm </td><td> 0.5M </td></tr><tr><td> PMSF </td><td> 200 mL </td><td></td><td></td></tr><tr><td> Completa inibitore della proteasi mini </td><td> 1 compressa </td><td></td><td></td></tr><tr><td> Acqua </td><td> QSP </td><td></td><td></td></tr></table> Precipitazioni buffer: <table border="1"><tr><td></td><td> 100 ml </td><td> Concentrazione finale </td></tr><tr><td> TCA </td><td> 20 mL </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> Tampone di lavaggio: <table border="1"><tr><td></td><td> 100 ml </td><td> Concentrazione finale </td></tr><tr><td> DTT </td><td> 0,1 ml </td><td> 0,1% </td></tr><tr><td> Aceton </td><td> 100 ml </td><td></td></tr></table> Etichettatura Buffer (conservare a -18 ° C in piccole aliquote): <table border="1"><tr><td></td><td> 1 ml </td><td> Concentrazione finale </td></tr><tr><td> Urea </td><td> 140 mg </td><td> 7M </td></tr><tr><td> Tiourea </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 microlitri </td><td> 30 mM </td></tr><tr><td> Acqua </td><td> 1 ml </td><td></td></tr></table>

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

Fusaria funghi filamentosi sono in grado di produrre tossine diverse. Micotossine del genere Fusarium, quali il deossinivalenolo, nivalenolo, T2, zearelenone, fusaric acido, moniliformina, ecc .. avere effetti nocivi sulla salute umana e animale e alcuni sono considerati come fattori di patogenicità. Studi di proteomica hanno mostrato di essere efficace per decifrare i meccanismi di produzione di tossine (Taylor et al., 2008) così come per identificare i fattori patogeni potenziali (Carta et al., 2007, Houterman et al., 2007) in Fusaria. Diventa quindi fondamentale stabilire metodi affidabili per il confronto tra gli studi di proteomica al fine di contare su vere differenze si trovano in espressione proteica tra esperimenti, ceppi e laboratori. La procedura che verrà descritta dovrebbe contribuire ad un aumento del livello di standardizzazione delle procedure di proteomica in due modi. Il protocollo girato è usato per aumentare il livello di dettagli che possono essere descritti con precisione. Inoltre, la disponibilità di procedure standardizzate di processo biologico replica dovrebbe garantire una robustezza maggiore di dati, tenendo conto anche del fattore umano all&#39;interno della riproducibilità tecnica della procedura di estrazione. Il protocollo descritto richiede 16 giorni per il suo completamento: quattordici giorni per le culture e due giorni per l&#39;estrazione di proteine ​​(figura 1). In breve, i ceppi di Fusarium sono coltivate su terreni solidi per 4 giorni, sono poi manualmente frammentato e trasferito in un media tossina modificata indurre (Jiao et al, 2008.) Per 10 giorni. Micelio viene raccolto per filtrazione attraverso uno strato Miracloth. Rettifica è eseguita in una camera fredda. Diversi operatori effettuato l&#39;estrazione repliche (n = 3) al fine di tener conto della distorsione dovuta a varianti tecniche (figura 2). L&#39;estrazione è basata su un SDS / DTT tampone, come descritto nella Taylor et al. (2008) con lievi modifiche. Estrazione di proteine ​​totali ha richiesto un processo di precipitazione delle proteine ​​con Aceton / TCA / DTT lavaggio del buffer durante la notte e acetone / DTT (figura 3a, 3b). Proteine ​​sono state finalmente resolubilized nella proteina di etichettatura buffer e quantificati. I risultati dell&#39;estrazione sono stati visualizzati su un gel 1D (Figura 4, SDS-PAGE), prima di procedere alla 2D gel (IEF / SDS-PAGE). La stessa procedura può essere applicata per analisi proteomica su altri substrati di coltivazione e di altri funghi filamentosi (Miles et al., 2007).

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

Estrazione di proteine ​​per l&#39;analisi proteomica di specie fungine richiede alti livelli di standardizzazione da realizzare in accordo con le informazioni minime su un esperimento di proteomica (MIAPE) le linee guida. Vi presentiamo un video-protocollo che prevede una procedura di minimizzazione pregiudizi sperimentale durante l&#39;induzione della tossina e l&#39;estrazione di proteine ​​da<em&gt; Fusarium spp.</em

Tossina induzione ed estrazione di proteine ​​da Fusarium Spp. Culture per studi di proteomica

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

Research

JoVE Journal

Biology

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

16.9K Views.  Centre de Recherche Public-Gabriel Lippmann. Estrazione di proteine ​​per l'analisi proteomica di specie fungine richiede alti livelli di standardizzazione da realizzare in accordo con le informazioni minime su un esperimento di proteomica (MIAPE) le linee guida. Vi presentiamo un video-protocollo che prevede una procedura di minimizzazione pregiudizi sperimentale durante l'induzione della tossina e l'estrazione di proteine ​​da Fusarium spp.

Video: 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-dissezione del cervello di ratto per estrazione RNA o proteine ​​da Regione specifiche del cervello

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

Ricerca

Biologia

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).

Separazione elettroforetica delle proteine

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.

Intervista: Protein Folding e lo studio di malattie neurodegenerative

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.

Preparazione delle Culture del cervello di ratto aggregati per Neuron e Studi sullo Sviluppo Glia

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.

Microfluidica digitale per trattamento automatizzato di proteomica

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.

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

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.

Estrazione DNA da materiale in paraffina per le analisi genetiche ed epigenetiche

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.

Sviluppo sessuale e scarico in ascospore Fusarium graminearum</em

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

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.

Metabolic Etichettatura e con membrana frazionamento per proteomica comparativa Analisi del<em&gt; Arabidopsis thaliana</em&gt; Culture sospensione cellulare

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

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preparazione del campione proteomica dalla fissati in formalina e incluso in paraffina Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Tossina induzione ed estrazione di proteine da Fusarium Spp. Culture per studi di proteomica

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Estrazione DNA da materiale in paraffina per le analisi genetiche ed epigenetiche

Sviluppo sessuale e scarico in ascospore Fusarium graminearum

Metabolic Etichettatura e con membrana frazionamento per proteomica comparativa Analisi del

Preparazione del campione proteomica dalla fissati in formalina e incluso in paraffina Tissue

Tossina induzione ed estrazione di proteine ​​da Fusarium Spp. Culture per studi di proteomica

Riepilogo

Esplora Altri Video

Capitoli in questo video

Micro-dissezione del cervello di ratto per estrazione RNA o proteine ​​da Regione specifiche del cervello

Separazione elettroforetica delle proteine

Intervista: Protein Folding e lo studio di malattie neurodegenerative

Preparazione delle Culture del cervello di ratto aggregati per Neuron e Studi sullo Sviluppo Glia

Microfluidica digitale per trattamento automatizzato di proteomica

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

Estrazione DNA da materiale in paraffina per le analisi genetiche ed epigenetiche

Sviluppo sessuale e scarico in ascospore Fusarium graminearum

Metabolic Etichettatura e con membrana frazionamento per proteomica comparativa Analisi del

Preparazione del campione proteomica dalla fissati in formalina e incluso in paraffina Tissue

Tossina induzione ed estrazione di proteine da Fusarium Spp. Culture per studi di proteomica

Micro-dissezione del cervello di ratto per estrazione RNA o proteine da Regione specifiche del cervello