我们感谢他们的技术贡献Servane Contal和鲍里斯Untereiner。我们承认FNR的“FUTOX”项目的支持。

该协议要求16天完成。详细的时间表是在图1。 缓冲液配制的详细信息<ul><li>制备细胞裂解液（每个样品3ML）。 </li><li>制备的洗涤液（50毫升每个样品）。这个缓冲区应预先冷冻和储存在-20 ° C在冰箱，直到进一步的分析，并在整个过程中保持在冰上。 </li><li>制备沉淀的缓冲液（每个样品20ML）。这个缓冲区应预先冷冻和储存在冰箱，直到需要在整个过程中，冰在-20 ° C。 </li><li>应当优先进行的工作，进入冷室在4 ° C。 </li></ul>真菌种类用于蛋白质组学分析三株被列为其形态特征和翻译延伸因子-α- 1分析的基础上禾谷镰刀菌（奥唐奈等， 1998） 这些菌株属于CRP -加布里埃尔李普曼收集，维护和存放在 - 80℃在15％甘油。 菌丝体的制备真菌培养4天，在25 ° C交替光明与黑暗的时期每12小时在V8上。文化支离破碎，用消毒的刀片和这些文化被用来接种250毫升锥形瓶含有100毫升的毒素诱导媒体件。收获后10天菌丝体，并从中分离，用​​无菌过滤器的过滤文化。菌丝体均用无菌水冲洗3次，以去除介质的残留物和其他化合物。我们然后立即加入液氮冻结的细胞，保持样品在-80 ° C。 蛋白提取的一般描述使用在图3a和3b中所描述的方法提取蛋白样品。 镰刀菌的样品在液氮研磨的粉末收集在10毫升聚四氟乙烯管。样品与裂解液孵育30分钟，煮沸10分钟，离心2次，在室温（15分钟12000克）。收集上清和沉淀缓冲液孵育过夜在-20 ° C。后离心4 ° C30000克45分钟，沉淀蛋白质的沉淀物洗净，用冷丙酮含数码地面电视的3倍。最后，沉淀空气干燥和蛋白质resolubilized标签缓冲区中，调整pH值至8。 30毫升的子样本被删除总蛋白定量和蛋白电泳在-20 ° C保存，直到剩余的上清液。 <img src="/files/ftp_upload/1690/1690fig1.jpg" alt="图1" /> 图1。时间轴的过程。 <img src="/files/ftp_upload/1690/1690fig2.jpg" alt="图2" /> 图2样品处理多个运营商的计划。 <img src="/files/ftp_upload/1690/1690fig3atmb.jpg" alt="图3a" border="0" /> 图3a。 <img src="/files/ftp_upload/1690/1690fig3btmb.jpg" alt="图3b" border="0" /> 图3b，请<a href="/files/ftp_upload/1690/1690fig3a.jpg">点击这里</a>看到的图3A更大的版本，或<a href="/files/ftp_upload/1690/1690fig3b.jpg">在</a>这里为图3b更大的版本。 图3a - B蛋白的提取过程的流程图（A：第一天，B：第二天） 。 <img src="/files/ftp_upload/1690/1690fig4tmb.jpg" alt="图4" border="0" /> 图4。蛋白提取物的一维图像。蛋白的运行，直到蓝色熔岩紫染色的凝胶年底完成迁移。图4看到一个更大的版本， <a href="/files/ftp_upload/1690/1690fig4.jpg">请点击</a>这里。

<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>

使用这种技术（Kim 等人 ，2007年审查）的出版物的数量增加导致真菌生物学领域内的蛋白质组学方法的近期利益。依靠蛋白的提取方法提取程序的组合，他们往往是很难描述在方法论部分和需要的专业知识，在处理潜在的故障排除方法。 至于其他“组学”的办法，设置样品和数据处理的标准是必要的，才能产生可​​靠的科学信息。此外，样品和操作程序应得到充分的描述，以允许共享不同的“组学”实验结果的解释。跨数据挖掘在基因组学，蛋白质组学，代谢组学，充分利用潜力，这将是必不可少的... （莫里森等人 ，2006年）。约蛋白质组学实验的最低限度的信息已经起草工作组已成立，以解决一个实验的所有方面（凝胶电泳，质谱，分子间的相互作用，蛋白质修饰，蛋白质组信息，来样加工）。目前没有明确的信息，来样加工程序进行。由于蛋白的提取程序，在确定蛋白质组研究的最终质量，以实施程序，可以增加MIAPE指导方针的框架内的标准化（Taylor等，2007）的重要性，我们提出了使用视频协议详述样品处理。影片描述的实验可能会作出重大贡献的来样加工的信息数量增加，可能会导致更好的“组学”实验（Pasquali，2007年）的可重复性。 事实上，整个实验室的可重复性是一项基本要求，为了保证蛋白质组学研究结果（http://www.fixingproteomics.org）的有效性。跨实验室的可重复性是由两个因素：不同的仪器和不同的运营商操纵样品的影响。 在描述的协议，我们建议执行生物复制涉及多个运营商增加（图2和视频）数据（即技术变化的结果是考虑到）的可靠性。 蛋白提取描述的过程是基于SDS和暖气。这是以前，以保证良好的纯度和蛋白质的数量（1996年桥）。 SDS的耦合，用沸水溶解细胞壁，疏水蛋白，防止形成的低聚物，中止蛋白质沉淀。沸点还允许蛋白酶失活。 EDTA，PMSF和完整的迷你蛋白酶抑制剂也产生同样的效果。数码地面电视的消除和促进蛋白增溶蛋白质之间的二硫键。 为了消除前IEF SDS，蛋白质沉淀丙酮/ TCA /数码地面电视。此外，丙酮/ TCA /数码地面电视的沉淀可以去除一些污染物，如脂类，核酸，盐类或/和酚类化合物，这些化合物存在时。 SDS的一样，这些分子可以防止在国际盛事基金的一个很好的迁移。这种沉淀，这是必要的丙酮/数码地面电视，洗沉淀的蛋白质，以去除TCA的，因为它可以干扰IEF。 一个良好的样品制备是好的结果的关键。对于这一点，它也是必不可少的，以避免污染环境工作无粉手套和尊重良好实验室规范的蛋白质。从技术角度来看，在所描述的协议，获得足够数量和纯度蛋白质最关键的步骤是分两个阶段。首先，研磨阶段，完整的细胞破坏是根本释放的蛋白质;和第二，总蛋白的纯化，一个阶段，包括取消大多数脂类，DNA和其它污染物，可能会干扰蛋白质的迁移。

媒体和解决方案 掌上电脑 ：39克马铃薯葡萄糖琼脂（Difco公司）; 1升蒸馏水。 V8琼脂 ：200毫升V8（坎贝尔，美国），2G碳酸钙（Sigma公司），16G琼脂（DIFCO），800毫升蒸馏水 毒素诱导媒体 ：1G K 2 HPO 4，0.5克氯化钾，0.5克硫酸镁4 7H 2 O，10毫克FeEDTA，2G大号谷氨酸，蒸馏水1L 10G蔗糖。 裂解缓冲液 ： <table border="1"><tr><td></td><td> 100毫升</td><td>终浓度</td><td></td></tr><tr><td>三，盐酸PH8 </td><td> 5ML </td><td> 50MM </td><td> 1M </td></tr><tr><td> SDS </td><td> 2克或10ml </td><td> 2％ </td><td> 20％ </td></tr><tr><td>数码地面电视</td><td> 500毫升</td><td> 10MM </td><td> 2M </td></tr><tr><td> EDTA </td><td> 20ML </td><td> 0.1MM </td><td> 0.5M </td></tr><tr><td> PMSF </td><td> 200毫升</td><td></td><td></td></tr><tr><td>完整的迷你蛋白酶抑制剂</td><td> 1片</td><td></td><td></td></tr><tr><td>水</td><td> QSP </td><td></td><td></td></tr></table> 沉淀缓冲液 ： <table border="1"><tr><td></td><td> 100毫升</td><td>终浓度</td></tr><tr><td>三氯乙酸</td><td> 20ML </td><td> 20％ </td></tr><tr><td>数码地面电视</td><td> 0.1ml的</td><td> 0.1％ </td></tr><tr><td>丙酮</td><td> QSP </td><td></td></tr></table> 洗涤缓冲液： <table border="1"><tr><td></td><td> 100毫升</td><td>终浓度</td></tr><tr><td>数码地面电视</td><td> 0.1ml的</td><td> 0.1％ </td></tr><tr><td>丙酮</td><td> 100毫升</td><td></td></tr></table> 标签缓冲区（储存在-18 ° C的小等分）： <table border="1"><tr><td></td><td> 1ML </td><td>终浓度</td></tr><tr><td>尿素</td><td> 140毫克</td><td> 7M </td></tr><tr><td>硫脲</td><td> 50毫克</td><td> 2M </td></tr><tr><td> CHAPS </td><td> 40毫克</td><td> 4％ </td></tr><tr><td>三</td><td> 30μL </td><td> 30毫米</td></tr><tr><td>水</td><td> 1毫升</td><td></td></tr></table>

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

Fusaria是丝状真菌能够产生不同的毒素。镰刀霉菌毒素，如呕吐毒素，nivalenol，T2，zearelenone，镰刀菌酸moniliformin，等..对人类和动物健康有不良影响，有些是致病因素考虑。蛋白质组学研究表明，要有效地破译毒素生产机制（Taylor等，2008），以及确定潜在的致病因素（纸张等人，2007 年，Houterman等，2007） ，在Fusaria。因此，它成为根本，以建立可靠的方法之间的比较蛋白质组学研究，以依靠蛋白表达实验，菌种和实验室之间的真正差异。将要描述的过程应该通过两种方式蛋白质组学程序的标准化水平的提高作出贡献。拍过的协议是用来增加可以精确地描述的细节水平。此外，标准化的程序来处理生物可用性，复制，应保证较高的鲁棒性的数据，同时也考虑到内的提取过程中的技术可重复性的人为因素。 描述该协议要求16天完成14天的文化和两天蛋白的提取（图1）。 简单地说， 镰刀菌菌株4天的固体培养基上生长，然后手动割裂为10天，并转移到一个修改后的毒素诱导媒体（ 焦等，2008）。。通过Miracloth层过滤收集菌丝。在冷室中进行研磨。不同的运营商进行提取，复制（N = 3），以便考虑到由于技术的变化（图2）偏置。提取是基于Taylor等人所描述的SDS /数码地面电视的缓冲区。 （2008年），稍作修改。总蛋白的提取所需的使用丙酮/ TCA / DTT缓冲区隔夜和丙酮/数码地面电视洗衣机（图3A，3B）的蛋白质的降水过程。终于resolubilized蛋白在蛋白标签缓冲区和量化。提取的结果是一维凝胶（图4 SDS - PAGE），然后再进行二维凝胶电泳（IEF / SDS - PAGE）。同样的过程可以应用于其他越来越多的媒体和其他丝状真菌（ 万里等人，2007年）的蛋白质组分析。

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

在真菌的蛋白质组分析蛋白质的提取要求高的水平要完成标准化，按照有关一个蛋白质组学实验（MIAPE）指引的最低限度的信息。我们目前的视频协议，其中包括一个程序，最大限度地减少在毒素诱导和蛋白提取实验偏见<em&gt;镰刀菌。</em

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

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 次观看.  Centre de Recherche Public-Gabriel Lippmann. 在真菌的蛋白质组分析蛋白质的提取要求高的水平要完成标准化，按照有关一个蛋白质组学实验（MIAPE）指引的最低限度的信息。我们目前的视频协议，其中包括一个程序，最大限度地减少在毒素诱导和蛋白提取实验偏见镰刀菌。

视频: 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.

从特定的大脑区域的RNA或蛋白质提取大鼠脑微解剖

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

科研

生物学

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).

电泳分离蛋白质

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.

专访：蛋白质折叠与神经退行性疾病的研究

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.

制备大鼠脑组织神经元和神经胶质发展研究总结文化

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.

数字微流体进行自动蛋白质组学处理

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.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

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.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

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.

性发展和有性放电禾谷镰刀菌</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.

天真成人干细胞的分离从初级人类成纤维细胞培养

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.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

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

毒素诱导和蛋白质提取镰刀 SPP。文化