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

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 ...

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 ...

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 ...

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.

Proteómica Preparación de muestras de formol fija y embebidos en parafina de tejidos

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

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

La extracción del veneno y veneno glándula microdisecciones de arañas de Proteómica y análisis Transcriptomic

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Proteína entrega directa de células de mamíferos Utilización de Células permeable Cys<sub&gt; 2</sub&gt; -Su<sub&gt; 2</sub&gt; Dominios de zinc-finger

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...