Damos las gracias a Servane Contal y Untereiner Boris por su contribución técnica. Queremos agradecer el apoyo del FNR &quot;FUTOX&quot; del proyecto.

El protocolo requiere 16 días para su finalización. El plazo se detalla en la figura 1. Detalles para la preparación de buffer <ul><li> Preparación de la solución amortiguadora de lisis (3 ml por muestra). </li><li> Preparación del tampón de lavado (50 ml por muestra). Este buffer debe ser pre-enfriado y almacenado a -20 ° C en el congelador hasta su posterior análisis y se mantuvo en hielo durante todo el procedimiento. </li><li> Preparación del tampón de precipitación (20 ml por muestra). Este buffer debe ser pre-enfriado y almacenado a -20 ° C en el congelador hasta que se necesite y se mantuvo en hielo durante todo el procedimiento. </li><li> El trabajo preferentemente deben llevarse a cabo en la cámara de frío a 4 º C. </li></ul> Especies de hongos Tres cepas utilizadas para los análisis proteómicos se clasificaron a Fusarium graminearum sobre la base de sus características morfológicas y traducción-Alargamiento de alfa-1 factor de análisis (O&#39;Donnell et al., 1998) Estas cepas pertenecen a la colección de PCR-Gabriel Lippmann y se mantuvieron y se almacena a - 80 ° C en el 15% de glicerol. Preparación de los micelios Los hongos se cultivaron en V8 de 4 días a 25 ° C alternando período de luz y la oscuridad cada 12 horas. Los cultivos fueron fragmentadas con una cuchilla estéril y piezas de estas culturas se utilizaron para inocular 250 ml frascos erlenmeyer que contiene 100 ml de medio de inducción de la toxina. Micelios fueron cosechadas después de 10 días y fueron separados de filtrado por medio de la cultura con un filtro estéril. Micelio se lavaron 3 veces con agua estéril para eliminar los residuos de media y otros compuestos. A continuación, inmediatamente se congelaron las células mediante la adición de nitrógeno líquido y se mantienen las muestras a -80 ° C. Descripción general de la extracción de proteínas Las muestras de proteínas se obtuvieron mediante el método descrito en la figura 3A y 3B. Las muestras de Fusarium se molieron en nitrógeno líquido y el polvo fue recogido en tubos de 10 ml de teflón. Las muestras fueron incubadas 30 minutos con el tampón de lisis, se hierve durante 10 minutos, y se centrifuga dos veces a temperatura ambiente (12000g durante 15 minutos). Se recogieron los sobrenadantes y se incuba a 20 ° C con tampón de la noche. Después de la centrifugación a 4 ° C Comprar 30000G durante 45 minutos, los pellets de las proteínas precipitadas se lavaron 3 veces con acetona fría que contiene la TDT. Finalmente, los sedimentos se secaron al aire y las proteínas se resolubilizado en el buffer de etiquetado, para ajustar el pH a 8. Una sub-muestra de 30 ml fue eliminado debido a que la cuantificación de proteínas totales y el resto de sobrenadante se almacenó a -20 ° C hasta la electroforesis de proteínas. <img src="/files/ftp_upload/1690/1690fig1.jpg" alt="Figura 1" /> Figura 1. Cronología del procedimiento. <img src="/files/ftp_upload/1690/1690fig2.jpg" alt="Figura 2" /> Figura 2. Sistema de múltiples operadores de procesamiento de muestras. <a href="/files/ftp_upload/1690/1690fig3a.jpg"><img src="/files/ftp_upload/1690/1690fig3atmb.jpg" alt="Figura 3a" border="0" /></a> Figura 3a. <a href="/files/ftp_upload/1690/1690fig3b.jpg"><img src="/files/ftp_upload/1690/1690fig3btmb.jpg" alt="Figura 3b" border="0" /></a> Figura 3b. Las figuras 3a-b Los diagramas de flujo para el procedimiento de extracción de proteínas (A: primer día; B: segundo día).. <a href="/files/ftp_upload/1690/1690fig4.jpg"><img src="/files/ftp_upload/1690/1690fig4tmb.jpg" alt="Figura 4" border="0" /></a> Figura 4. 1D imagen de extractos de proteínas. Las proteínas se extenderá hasta la migración completa de azul a la final del gel fue teñido con púrpura lava.

<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>

El reciente interés por la proteómica en el dominio de la biología de hongos llevó a un mayor número de publicaciones que utilizan esta técnica (que se examinan en Kim et al., 2007). Métodos para la extracción de proteínas se basan en una combinación de procedimientos de extracción, a menudo son apenas descritos en las secciones metodológicas y requiere experiencia en el trato con los métodos de solución de problemas potenciales. En cuanto a otras &quot;ómicas&quot; enfoques, el establecimiento de normas para el procesamiento de muestras y de datos es esencial a fin de generar información científica confiable. Además las muestras y los procedimientos de manipulación deben ser adecuadamente descrito con el fin de permitir una interpretación compartida entre los diferentes resultados &quot;ómicas&quot; experimentos. Esto sería esencial para explotar plenamente las potencialidades de la cruzada de minería de datos en genómica, proteómica, metabolómica, ... (Morrison et al., 2006). Información mínima sobre un experimento de proteómica se ha elaborado y grupos de trabajo se han establecido con el fin de gestionar todos los aspectos de un experimento (electroforesis en gel, la espectrometría de masas, las interacciones moleculares, proteínas modificaciones, Informática Proteómica, muestra la transformación). Por el momento no hay información definida están disponibles en los procedimientos de procesamiento de las muestras. Dada la importancia de los procedimientos de extracción de proteínas en la determinación de la calidad final de los estudios proteómicos, con el fin de poner en práctica procedimientos que pueden aumentar la normalización en el marco de las directrices MIAPE (Taylor et al., 2007), propuso el uso de un protocolo de video que detalla la muestra procesamiento. Descripción de vídeo de los experimentos pueden contribuir sustancialmente a aumentar la cantidad de información sobre el procesamiento de muestras que pueden resultar en una mejor reproductibilidad de &quot;ómicas&quot; experimentos (Pasquali, 2007). De hecho, la reproducibilidad entre laboratorios es un requisito fundamental para garantizar la validez de los resultados de la proteómica (http://www.fixingproteomics.org). La reproducibilidad cruzada de laboratorio está influida por dos factores: diferentes instrumentaciones y la manipulación de las muestras de los diferentes operadores. En el protocolo descrito, se propone llevar a cabo réplicas biológicas participación de operadores múltiples (Figura 2 y de vídeo) para aumentar la fiabilidad de los datos (es decir, la variación técnica de los resultados se tiene en cuenta). El procedimiento descrito para la extracción de la proteína se basa en la SDS y calefacción. Esto se había demostrado que garantizan una buena pureza y la cantidad de proteínas (Bridge, 1996). SDS, junto con hirviendo disuelve las paredes celulares, las proteínas hidrofóbicas y previene la formación de oligómeros que la precipitación de las proteínas de abortar. Ebullición permite también la inactivación de las proteasas. El mismo efecto se produce también por EDTA, PMSF y el inhibidor de la proteasa completa mini. TDT elimina puentes disulfuro entre las proteínas y en facilitar la solubilización de las proteínas. Con el fin de eliminar SDS antes de IEF, las proteínas son precipitadas con acetona / TCA / TDT. Por otra parte, la acetona / TCA / TDT precipitación permite la eliminación de algunos contaminantes, como los lípidos, ácidos nucleicos, sales y / o compuestos fenólicos cuando estos compuestos están presentes. Como el SDS, estas moléculas impiden una migración bien durante IEF. A raíz de esta precipitación, es necesario lavar las proteínas precipitadas con acetona / TDT, con el fin de eliminar el TCA, ya que puede interferir con el IEF. Una buena preparación de la muestra es la clave para obtener buenos resultados. Para ello, también es esencial para evitar la contaminación de las proteínas del ambiente de trabajo con los guantes sin polvo y respetando las buenas prácticas de laboratorio. Desde el punto de vista técnico, en el protocolo descrito, los pasos más importantes para la obtención de cantidades suficientes y la pureza de las proteínas de dos fases. En primer lugar, la fase de trituración en la completa destrucción de la célula es fundamental para la liberación de proteínas, y en segundo lugar, la purificación de proteínas totales, una fase que abarca la eliminación de la mayoría de los lípidos, ADN y otros contaminantes que puedan interferir con la migración de las proteínas.

Los medios de comunicación y soluciones PDA: 39 g de papa dextrosa agar (Difco), 1 l de agua destilada. Agar V8: 200 ml V8 (Campbell, EE.UU.), 2 g de CaCO3 (Sigma), Agar 16g (DIFCO), 800 ml de agua destilada Los medios de comunicación la toxina induce: 1 g de K 2 HPO 4, 0,5 g KCl, 0,5 g MgSO 4 7H 2 O, 10 mg FeEDTA, 2 g de ácido glutámico L, sacarosa 10 g en 1 litro de agua destilada. Tampón de lisis: <table border="1"><tr><td></td><td> 100 ml </td><td> La concentración final </td><td></td></tr><tr><td> Tris-HCl pH 8 </td><td> 5 ml </td><td> 50 mM </td><td> 1M </td></tr><tr><td> SDS </td><td> 2g o 10 ml </td><td> 2% </td><td> 20% </td></tr><tr><td> TDT </td><td> 500 ml </td><td> 10 mM </td><td> 2M </td></tr><tr><td> EDTA </td><td> 20 ml </td><td> 0.1 mM </td><td> 0,5 M </td></tr><tr><td> PMSF </td><td> 200 ml </td><td></td><td></td></tr><tr><td> Inhibidor de la proteasa completa mini- </td><td> 1 comprimido </td><td></td><td></td></tr><tr><td> De agua </td><td> Csp </td><td></td><td></td></tr></table> Precipitación Buffer: <table border="1"><tr><td></td><td> 100 ml </td><td> La concentración final </td></tr><tr><td> TCA </td><td> 20 ml </td><td> 20% </td></tr><tr><td> TDT </td><td> 0,1 ml </td><td> 0,1% </td></tr><tr><td> Acetona </td><td> Csp </td><td></td></tr></table> Tampón de lavado: <table border="1"><tr><td></td><td> 100 ml </td><td> La concentración final </td></tr><tr><td> TDT </td><td> 0,1 ml </td><td> 0,1% </td></tr><tr><td> Acetona </td><td> 100 ml </td><td></td></tr></table> Etiquetado Buffer (almacén a -18 ° C en pequeñas alícuotas): <table border="1"><tr><td></td><td> 1 ml </td><td> La concentración final </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 l </td><td> 30 mM </td></tr><tr><td> De agua </td><td> 1 mL </td><td></td></tr></table>

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

Fusaria son hongos filamentosos capaces de producir toxinas diferentes. Micotoxinas de Fusarium como el deoxinivalenol, el nivalenol, T2, zearelenone, fusárico ácido, moniliformina, etc .. tener efectos adversos en la salud humana y animal y algunos son considerados como factores de patogenicidad. Estudios de proteómica se mostró eficaz para descifrar los mecanismos de producción de toxinas (Taylor et al., 2008), así como para identificar los posibles factores patogénicos (Documento et al., 2007, Houterman et al., 2007) en Fusaria. Se convierte, pues, fundamental para establecer métodos confiables para la comparación entre los estudios de proteómica con el fin de contar con verdaderas diferencias en la expresión de proteínas entre los experimentos, las cepas y los laboratorios. El procedimiento que se describe debe contribuir a un mayor nivel de estandarización de los procedimientos de proteómica de dos maneras. El protocolo filmado se utiliza para aumentar el nivel de detalles que pueden ser descritos con precisión. Por otra parte, la disponibilidad de procedimientos normalizados para los procesos biológicos replica debe garantizar una mayor solidez de los datos, teniendo en cuenta también el factor humano dentro de la reproductibilidad técnica del procedimiento de extracción. El protocolo descrito requiere 16 días para su ejecución: catorce días para que las culturas y dos días para la extracción de proteína (figura 1). En resumen, las cepas de Fusarium son cultivadas en medios sólidos durante 4 días, que son luego manualmente fragmentada y se transfieren a un medio de inducción de la toxina modificada (Jiao et al, 2008.) Durante 10 días. Micelio se recoge por filtración a través de una capa de Miracloth. La molienda se realiza en una cámara de frío. Diferentes operadores realizaron la extracción de repeticiones (n = 3) con el fin de tener en cuenta el sesgo debido a las variaciones técnicas (figura 2). La extracción se basa en un buffer SDS / DTT como se describe en Taylor et al. (2008) con ligeras modificaciones. Extracción de la proteína total requerido un proceso de precipitación de las proteínas con acetona / TCA / TDT tampón de lavado durante la noche y acetona / TDT (figura 3a, 3b). Las proteínas fueron resolubilizado finalmente en el buffer de las proteínas de etiquetado y cuantificados. Los resultados de la extracción se visualizaron en un gel de 1D (Figura 4, SDS-PAGE), antes de proceder a geles 2D (IEF / SDS-PAGE). El mismo procedimiento puede ser aplicado para el análisis proteómico de otros medios de cultivo y otros hongos filamentosos (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

Extracción de proteínas de los análisis proteómicos de las especies de hongos requiere un alto nivel de estandarización que se realiza de acuerdo con la información mínima sobre un experimento de proteómica (MIAPE) directrices. Se presenta un video-protocolo que incluye un procedimiento para disminuir el sesgo experimental durante la inducción de la toxina y la extracción de proteínas a partir de<em&gt; Fusarium spp.</em

La toxina de inducción y extracción de proteínas a partir de Fusarium Spp. Los cultivos para los estudios proteómicos

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 vistas.  Centre de Recherche Public-Gabriel Lippmann. Extracción de proteínas de los análisis proteómicos de las especies de hongos requiere un alto nivel de estandarización que se realiza de acuerdo con la información mínima sobre un experimento de proteómica (MIAPE) directrices. Se presenta un video-protocolo que incluye un procedimiento para disminuir el sesgo experimental durante la inducción de la toxina y la extracción de proteínas a partir de 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-disección de cerebro de rata para el ARN o la extracción de proteínas a partir de región específica del cerebro

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

Investigación

Biología

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).

Separación electroforética de las proteínas

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.

Entrevista: plegamiento de proteínas y Estudios de Enfermedades Neurodegenerativas

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.

Preparación de las Culturas Rata total del cerebro de las neuronas y la glía de Estudios para el Desarrollo

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.

Microfluídica digitales para el procesamiento automatizado de Proteómica

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.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

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.

Extracción de ADN a partir de material incluido en parafina para análisis genéticos y epigenéticos

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.

Desarrollo Sexual y descarga en las ascosporas 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.

Naïve Adult Stem Cells Aislamiento de Primaria cultivos de fibroblastos humanos

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.

Metabólicas etiquetado y de membrana de fraccionamiento para Comparativo Proteómica Análisis de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensión celular

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