Agradecemos a Servane Contal e Boris Untereiner por sua contribuição técnica. Agradecemos o apoio da FNR &quot;FUTOX&quot; do projeto.

O protocolo exige que 16 dias para sua conclusão. O prazo é detalhado na figura 1. Detalhes para a preparação de buffer <ul><li> Preparação do tampão de lise (3mL por amostra). </li><li> Preparação do tampão de lavagem (50 ml por amostra). Esta reserva deve ser pré-refrigerados e armazenados a -20 ° C no freezer até análise mais aprofundada e mantidos em gelo durante todo o procedimento. </li><li> Preparação do tampão de precipitação (20 mL por amostra). Esta reserva deve ser pré-refrigerados e armazenados a -20 ° C no freezer até que seja necessário e mantidos em gelo durante todo o procedimento. </li><li> O trabalho deve preferencialmente ser realizado em câmara fria a 4 ° C. </li></ul> Espécies de fungos Três cepas utilizadas para análises proteômicas foram classificadas a Fusarium graminearum, com base em suas características morfológicas e Tradução Alongamento-Alpha-1 fator de análise (O&#39;Donnell et al., 1998) Essas cepas pertenciam à coleção Lippmann CRP-Gabriel e foram mantidos e armazenados a - 80 ° C em 15% de glicerol. Preparação dos micélios Fungos foram cultivados em V8 de 4 dias a 25 ° C alternadas período de claro e escuro a cada 12 hrs. Culturas foram fragmentados utilizando uma lâmina estéril e peças dessas culturas foram utilizadas para inocular balões de 250 ml Erlenmeyer contendo 100 mL da toxina induzindo-media. Micélios foram colhidas após 10 dias e foram separados do meio por filtração a cultura com um filtro estéril. Micélios foram lavadas 3 vezes com água estéril para remover resíduos de médio e outros compostos. Em seguida, imediatamente congelou as células pela adição de nitrogênio líquido e mantidas as amostras a -80 ° C. Descrição geral da extração de proteínas Amostras de proteínas foram extraídas com o método descrito na figura 3A e 3B. As amostras de Fusarium foram moídas em nitrogênio líquido eo pó foram recolhidos em 10 ml tubos de Teflon. As amostras foram então incubadas 30 minutos com o tampão de lise, cozidos por 10 minutos, e centrifugado duas vezes a temperatura ambiente (12000g por 15 minutos). Os sobrenadantes foram coletados e incubados a -20 ° C com tampão de precipitação durante a noite. Após centrifugação a 4 ° C 30000g por 45 minutos, as bolinhas das proteínas precipitadas foram lavadas três vezes com acetona gelada contendo DTT. Finalmente, as pelotas foram secas ao ar e as proteínas foram ressolubilizam no buffer de rotulagem, ajustando o pH a 8. A sub-amostra de 30 ml foi removido para a quantificação de proteínas totais eo sobrenadante restante foi armazenado a -20 ° C até eletroforese de proteínas. <img src="/files/ftp_upload/1690/1690fig1.jpg" alt="Figura 1" /> Figura 1. Timeline do procedimento. <img src="/files/ftp_upload/1690/1690fig2.jpg" alt="Figura 2" /> Figura 2. Esquema para múltiplos operadores de processamento de amostra. <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. Por favor, <a href="/files/ftp_upload/1690/1690fig3a.jpg">clique aqui</a> para ver uma versão ampliada da figura 3a, ou <a href="/files/ftp_upload/1690/1690fig3b.jpg">aqui</a> para uma versão ampliada da figura 3b. Figuras 3a-b Fluxogramas para o procedimento de extração de proteínas (A: primeiro dia; B: segundo dia).. <img src="/files/ftp_upload/1690/1690fig4tmb.jpg" alt="Figura 4" border="0" /> Figura 4. 1D imagem de extratos de proteína. Proteínas foram executados até a migração completa do azul para o final do gel que foi manchada com roxo lava. Por favor, <a href="/files/ftp_upload/1690/1690fig4.jpg">clique aqui</a> para ver uma versão ampliada da 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 em abordagens proteômicas no domínio da biologia de fungos levou a um aumento do número de publicações com essa técnica (como revistas em Kim et al., 2007). Métodos para a extração de proteínas dependem de uma combinação de procedimentos de extração, que são muitas vezes mal descritos nas seções metodológicas e requerem conhecimento especializado para lidar com os métodos de solução de problemas em potencial. Como para outros &quot;ómicas&quot; abordagens, estabelecendo normas para o processamento de amostras e dados é essencial a fim de gerar informações científicas confiáveis. Além disso as amostras e os procedimentos de manipulação devem ser adequadamente descritos de modo a permitir interpretação dos resultados compartilhados entre os diferentes &quot;ómicas&quot; experimentos. Isso seria essencial para a plena exploração do potencial cross-de mineração de dados em genômica, proteômica, metabolômica, ... (Morrison et al., 2006). Informações mínimas sobre um experimento proteômica foi elaborado e grupos de trabalho foram criados de forma a gerir todos os aspectos de uma experiência (de eletroforese em gel, Espectrometria de Massa, interações moleculares, modificações de proteínas, Informática Proteomics, o processamento da amostra). No momento nenhuma informação definida estão disponíveis em procedimentos de processamento da amostra. Dada a importância dos procedimentos de extração da proteína para determinar a qualidade final dos estudos de proteômica, a fim de implementar procedimentos que podem aumentar a padronização no âmbito das diretrizes MIAPE (Taylor et al., 2007), propusemos o uso de um protocolo de vídeo detalhando amostra processamento. Descrição do vídeo de experimentos pode contribuir substancialmente para aumentar o número de informações sobre o processamento da amostra, que pode resultar em melhor reprodutibilidade de &quot;ómicas&quot; experimentos (Pasquali, 2007). Na verdade, toda a reprodutibilidade laboratórios é um requisito fundamental para garantir a validade dos resultados proteômica (http://www.fixingproteomics.org). A reprodutibilidade cruz laboratório é influenciado por dois fatores: instrumentações diferentes e diferentes operadores manipulando as amostras. No protocolo descrito, propomos a realizar replica biológica envolvendo múltiplos operadores (Figura 2 e vídeo) para aumentar a confiabilidade dos dados (ou seja, variação da técnica dos resultados é levado em conta). O procedimento descrito para a extração de proteínas é baseado em SDS e aquecimento. Isto foi demonstrado anteriormente para garantir uma pureza e boa quantidade de proteínas (Bridge, 1996). SDS juntamente com ebulição dissolve as paredes das células, as proteínas hidrofóbicas e impede a formação de oligômeros que a precipitação de proteínas abortar. Ebulição permite também a inativação de proteases. O mesmo efeito é produzido também por EDTA, PMSF e inibidor da protease completa mini. TDT remove pontes dissulfeto e entre proteínas facilitando a solubilização da proteína. A fim de remover antes IEF SDS, as proteínas são precipitadas por acetona / TCA / TDT. Além disso, acetona / TCA / TDT precipitação permite remover alguns contaminantes, tais como lipídios, ácidos nucléicos, sais e / ou compostos fenólicos, quando estes compostos estão presentes. Como SDS, estas moléculas evitar uma migração bem durante IEF. Seguindo essa precipitação, é necessário lavar as proteínas precipitadas com acetona / DTT, a fim de remover TCA, pois ele pode interferir com IEF. A preparação da amostra boa é a chave para bons resultados. Para isso, é também essencial para evitar a contaminação de proteínas do ambiente de trabalho com luvas sem pó e respeitando as boas práticas laboratoriais. Do ponto de vista técnico, dentro do protocolo descrito, os passos mais críticos para obtenção de quantidades suficientes e pureza de proteínas são duas fases. Primeiro, a fase de moagem onde a destruição completa da célula é fundamental para a liberação de proteínas e, segundo, a purificação de proteínas totais, uma fase que engloba a remoção da maioria dos lipídios DNA, e outros contaminantes que possam interferir com a migração de proteínas.

Mídia e soluções PDA: 39 g Batata Dextrose Agar (Difco), 1 L de água destilada. V8 agar: 200 ml V8 (Campbell, EUA), 2g CaCO3 (Sigma), Agar 16g (DIFCO), 800 ml de água destilada Toxina mídia induzindo: 1g K 2 HPO 4, 0,5 g KCl, 0,5 g MgSO 4 7H 2 O, 10 mg FeEDTA, 2g L ácido glutâmico, sacarose 10g em 1L de água destilada. Tampão de Lise: <table border="1"><tr><td></td><td> 100mL </td><td> Concentração final </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 ou 10 ml </td><td> 2% </td><td> 20% </td></tr><tr><td> TDT </td><td> 500mL </td><td> 10mM </td><td> 2M </td></tr><tr><td> EDTA </td><td> 20mL </td><td> 0,1 mM </td><td> 0.5M </td></tr><tr><td> PMSF </td><td> 200mL </td><td></td><td></td></tr><tr><td> Inibidor da protease completa mini- </td><td> 1 comprimido </td><td></td><td></td></tr><tr><td> Água </td><td> Qsp </td><td></td><td></td></tr></table> Tampão de precipitação: <table border="1"><tr><td></td><td> 100mL </td><td> Concentração final </td></tr><tr><td> TCA </td><td> 20mL </td><td> 20% </td></tr><tr><td> TDT </td><td> 0,1 ml </td><td> 0,1% </td></tr><tr><td> Acetona </td><td> Qsp </td><td></td></tr></table> Lavar Buffer: <table border="1"><tr><td></td><td> 100mL </td><td> Concentração final </td></tr><tr><td> TDT </td><td> 0,1 ml </td><td> 0,1% </td></tr><tr><td> Acetona </td><td> 100mL </td><td></td></tr></table> Rotulagem de buffer (armazenamento a -18 ° C em alíquotas pequenas): <table border="1"><tr><td></td><td> 1mL </td><td> Concentração final </td></tr><tr><td> Uréia </td><td> 140 mg </td><td> 7M </td></tr><tr><td> Tiouréia </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 mL </td><td> 30 mM </td></tr><tr><td> Água </td><td> 1 mL </td><td></td></tr></table>

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

Fusaria são fungos filamentosos capazes de produzir toxinas diferentes. Micotoxinas Fusarium, como o desoxinivalenol, nivalenol, T2, zearelenone, ácido fusárico, moniliformin, etc .. ter efeitos adversos na saúde humana e animal e alguns são considerados como fatores de patogenicidade. Estudos de proteômica mostrou-se eficaz para decifrar os mecanismos de produção de toxinas (Taylor et al., 2008), bem como para a identificação de fatores patogênicos em potencial (Paper et al. 2007, Houterman et al., 2007) em Fusaria. Torna-se, portanto, fundamental para estabelecer métodos confiáveis ​​para a comparação entre os estudos de proteômica, a fim de contar com verdadeiras diferenças encontradas na expressão da proteína entre os experimentos, as cepas e laboratórios. O procedimento que será descrito deverá contribuir para um maior nível de padronização de procedimentos proteômica de duas maneiras. O protocolo filmado é usado para aumentar o nível de detalhes que pode ser descrito com precisão. Além disso, a disponibilidade de procedimentos padronizados para processo biológico replica deve garantir uma maior robustez dos dados, tendo em conta também o fator humano dentro da reprodutibilidade técnica do processo de extração. O protocolo descrito requer 16 dias para sua conclusão: 14 dias para as culturas e dois dias para a extração de proteínas (figura 1). Resumidamente, Fusarium cepas são cultivadas em meios sólidos por 4 dias, pois eles são, então, manualmente fragmentado e transferido para uma mídia toxina modificada indução (Jiao et al, 2008.) Por 10 dias. Micélio é coletado por filtração através de uma camada Miracloth. Moagem é realizada em uma câmara fria. Diferentes operadores realizada extração de repetições (n = 3), a fim de ter em conta o viés devido a variações técnica (figura 2). Extração foi baseado em um tampão SDS / TDT conforme descrito em Taylor et al. (2008) com ligeiras modificações. Extração de proteína total necessário um processo de precipitação das proteínas com acetona / TCA / TDT tampão de lavagem durante a noite e acetona / TDT (figura 3a, 3b). Proteínas foram finalmente ressolubilizam no buffer de proteína rotulagem e quantificados. Resultados da extração foram visualizados em gel 1D (Figura 4, SDS-PAGE), antes de prosseguir para 2D géis (IEF / SDS-PAGE). O mesmo procedimento pode ser aplicado para análises proteômicas em outros suportes de cultura e outros fungos 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

Extração de proteínas para análise proteômica em espécies de fungos requer um alto nível de padronização a ser realizado de acordo com as informações mínimas sobre um experimento proteômica (MIAPE) orientações. Nós apresentamos um vídeo protocolo que inclui um procedimento para minimizar viés experimental durante a indução da toxina e extração de proteínas de<em&gt; Fusarium spp.</em

Indução toxina e Extração de Proteínas de Fusarium Spp. Culturas de Estudos Proteômica

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 visualizações.  Centre de Recherche Public-Gabriel Lippmann. Extração de proteínas para análise proteômica em espécies de fungos requer um alto nível de padronização a ser realizado de acordo com as informações mínimas sobre um experimento proteômica (MIAPE) orientações. Nós apresentamos um vídeo protocolo que inclui um procedimento para minimizar viés experimental durante a indução da toxina e extração de proteínas de Fusarium spp.

Vídeo: 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-dissecção de cérebro de um rato para RNA ou Extração de Proteína da região específica do cérebro

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

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).

Separação eletroforética de 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: o dobramento de proteínas e Estudos de Doenças 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.

Preparação das Culturas cérebro de um rato Aggregate para Neuron Glia e Estudos de Desenvolvimento

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.

A microfluídica digital para processamento automatizado Proteômica

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.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery 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.

Extração de DNA de material parafinado Embedded para análises genéticas e epigenéticas

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.

Desenvolvimento Sexual e descarga de ascósporos em 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 isolamento de culturas primárias 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.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

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