这项工作是由Knut和Alice Wallenberg基金会赠款支持。整个人类蛋白质图集中心在乌普萨拉，斯德哥尔摩和印度的工作人员都承认他们的努力产生的人类蛋白质图集。作者希望拍戏时特别感谢的照片和忆念美Karlberg留言弗兰克·Hammar和SOFIE古斯塔夫森的援助援助与TMA的生产。

1。如何准备组织芯片生产组织（动画1） <ol><li>选择有关福尔马林固定石蜡包埋材料，包括相应的​​苏木染色组织切片（组织或细胞样本）。 </li><li>马克的组织切片上的相关领域。建议将新鲜切割的苏木染色部分相应的石蜡块。 </li><li> TMA的生产中使用的模板设计。随机样本，模板，以避免技术问题，如覆盖在整个TMA节和切片问题的抗体引起的工件内。添加额外的导向标志为导向的模板。 </li><li>组织的组织，根据模板。 </li></ol> 2。手动组织芯片制作（拍摄基本） <ol><li>为了能够获得标准化组织核心长度，标志着如管芯4.5毫米（ 图2）。 guidelINES为样本中心之间的间距： 核心规格0.6毫米 - 0.8-1.0毫米 核心大小1.0毫米 - 1.8-2.0毫米 核心规格1.5毫米 - 2.0-3.0毫米 核心规格2.0毫米 - 2.5-4.0毫米 建议离开2.5-3.0毫米利润率每一侧的石蜡块，石蜡，以避免开裂。 </li><li><ol class="lalpha"><li>装入选定要使用的拳。开始持有的地方冲六角螺丝松动。冲床正确定位时，在冲枢纽的凹槽坚决反对的金属棒放在塑料V型块。拧紧螺丝，确保边缘的金属夹子水平。 </li><li>收件人冲床（红色和绿色，分销商的具体标记）应放置到左边。捐助冲（绿色和蓝色，经销商具体标明），其中有一个稍大的直径应放置到正确的（ 图3）。 </li></ol></li><li><ol class="lalpha"><li>移动通过XY调节旋钮沿x和y轴的拳。正确调节旋钮沿x轴移动拳和左调节旋钮沿Y轴移动的拳。 两个调节旋钮上有一个数字千分尺读数。这些动旋钮时被激活。 </li><li> →按重置的ZERO / ABS键。 英寸和毫米之间的转变→按/毫米按钮。 孔之间的间距应为2.0毫米（测量孔中心之间），当使用一个9x8的模板1毫米​​打孔。 </li></ol></li><li><ol class="lalpha"><li>为了确保你不推入块一拳太远下来，击中录音带和破坏冲，放无石蜡的持有人的录像带，并设置收件人的冲床的底部位置1毫米以上的纸盒底部通过在深度停止螺丝左上角的Z-指南（ 图3）。 </li><li>在持有人把的收件人块和用最小的螺丝刀拧紧螺丝。不紧或拧紧螺丝，石蜡可能打破从纸盒宽松。 </li><li>收件人块推入收件人冲床下降约5毫米，并使用在冲手柄旋转冲头，来回一次（ 图3）。将块推入冲床时尽量下尽可能为5毫米深的洞。 </li><li>慢慢减轻向下推压，使弹簧可以移动一拳。使用管芯清空冲和丢弃的石蜡核心。 </li></ol></li><li>移动炮塔切换到捐助冲床（ 图3）。 </li><li>放置捐助块桥与超过收件人块持有人的捐助块（ 图3）。 </li><li><ol class="lalpha"><li>推动捐助puncH +进入捐助块选定区域。使用相应的苏木精和曙红染色幻灯片感兴趣的区域已标记为找到组织进行采样。手动举行捐助块，用左手的位置，并用右手推了一拳。请注意，你不应该管芯上推。深度停止不阻止拳打在适当的位置捐助块的议案，所以重要的是，你停止推动时的第二个标志是在管芯上可见。 </li><li>旋转手柄上冲一次（ 图3）。慢慢释放向下压力，同时仍保持对捐助块桥捐助块。 </li><li>捐助组织是最好砸出0.5毫米比收件人核心短。这一过程将确保在收件人的块面的内核可调对齐，避免宝贵的代表组织的损失。 </li></ol></li><li>拆除的桥梁和捐助块。使确保捐助冲床与早些时候作出的（4点）的孔对齐。你必须调整使用的固定螺钉，如果不对齐的捐助力度和在收件人块孔的打孔位置。 </li><li>小心推冲头向下，直到其尖端到达收件人块孔上方。持这种立场的同时，使用管芯流到洞的组织核心。核心表面应该是对齐的块面。 </li><li>移动炮塔后面的收件人冲。 </li><li>与XY调节旋钮，移动到下一个位置。 </li><li>从以上点4C重复，直到数组是完整的。 </li><li>当完成整个收件人块，松开在收件人块支架的螺钉。烤40分钟，42°C间的块。放在玻片上面临的切片面积的块。试管持有人（或其他适当的建设），并在T地点上放置块玻片他烤箱。烘烤后，从炉中取出试管支架和扁平块表面轻轻抚摸玻片。 </li><li>放置玻片上一个10分钟的冷却板块。 为了确保整个公会的代表组织，质量控制上每节50 日进行。 </li></ol> 3。如何节TMAs（动画） <ol><li>第传输系统（航模355S，MICROM）的使用切片机，切4微米厚的部分。该系统可用于所有当前的旋转切片机。该系统包括一个刀架与综合传输的桥梁，热水浴和一个控制单元。从刀片的部分通过转移到水浴桥湿转让表面上滑行。 </li><li>应采取的部分，从水浴后，他们立即伸出，避免敏感的组织点（如富含脂质，如乳腺癌和脑组织）的融化。解放军CE节上superfrost玻片。部分可以留在水中的时间取决于石蜡打蜡使用和水温。我们的实验室使用石蜡HistoLab产品AB，其中有一个熔点56-58℃，我们建议的水浴温度37-39°C。 </li><li>过夜干燥，在室温（RT），放置在一个幻灯片持有人的幻灯片。 </li><li>在50℃烘烤12-24小时的幻灯片。你有没有STS的运输如使用刷子部分手动从刀片水浴一个microtom。 </li></ol> 4。试验和免疫组织化学滴定法程序（动画3） 每个抗体是一个标准化的测试程序，使用专门设计TMAs包含组织和不同类型的细胞组合。其目的是评估个人和优化每一个抗体染色协议。最初稀释，基于蚂蚁ibody股票的主要抗体浓度进行测试。这个测试染色的成果用于指导进一步的稀释和协议的优化。 <ol><li> deparaffinization和二甲苯和梯度乙醇水化蒸馏水之前进行免疫。 </li><li>阻塞一步解渴内源性过氧化物酶的H 2 O 2 0.3％，95％乙醇5分钟。 </li><li>热诱导表面抗原检索（HIER）的执行中检索缓冲pH值6，使用压力锅炉（Decloaking室，医疗Biocare公司）作为热量来源为4分钟，在125°C。幻灯片完成后沸腾，保持中压锅炉，并让其冷却到90°C。为HIER总加工时间约45分钟。 </li><li>如果没有在最初的测试中取得决定性的结果是，做进一步的测试的基础上观测到的染色模式5 6。一项试验研究，在HPA高吞吐量的设置，显示其中HIER pH为6，但其他检索方法，如蛋白酶，微波炉和沸点较高或较低的pH值的缓冲预孵化，当然可以使用的最有用的抗原检索。通过改变免疫组化的条件，如改变抗体孵育时间，抗体稀释和二氨基联苯胺（DAB）的孵育时间，染色模式可以提供更确凿的结果​​。 </li></ol> 5。染色程序，Autostainer自动480（赛默飞世尔科技） 全部孵化完成后在室温下，此协议也可以用作手动设置在一个相同的试剂。 <ol><li>在洗涤缓冲液冲洗。 </li><li>超V型块的潜伏期为5分钟。 </li><li>在洗涤缓冲液冲洗。 </li><li>潜伏期30分钟的主要抗体。 </li><li>在洗涤缓冲液冲洗（X2）。 </li><li>孵育30分钟标记的辣根过氧化物酶聚合物。 </li><li>在洗涤缓冲液冲洗（X2）。 </li><li>在民建联发展的解决方案10分钟。 </li><li>在洗涤缓冲液冲洗（X2）。 </li><li>在迈耶斯苏木为5分钟* Counterstaining。当使用一个渐进的染色方法（例如：迈耶斯苏木）的力度取决于时间和没有分化是必需的。一种倒退的染色方法，如哈里斯苏木需要从组织中删除多余的染料，以突出结构的分化。 </li><li> *自来水冲洗。 </li><li>在碳酸锂水冲洗，经稀释的饱和溶液1分钟* 1:5。 </li><li>在自来水冲洗5分钟。 </li><li>幻灯片梯度乙醇脱水和幻灯片coverslipped（PERTEX，Histolab） </li></ol>适用于所有的试剂体积300μL每张幻灯片。 *步骤10-14在histostaining仪器（Autostainer自动XL，莱卡）和盖片是加在coverslipper系统（自动玻璃coverslipper的CV5030，徕卡）。 6。如何扫描幻灯片扫描幻灯片的方法是依赖于正在利用数字扫描仪，该协议只定义了如何使用自动扫描系统Aperio XT（Aperio技术）扫描幻灯片。在人类蛋白质图集设置20倍放大倍率的细胞组织和40X。自动扫描可能会遇到的焦点问题，由于异质性组织，包括在TMA不同组织的数量。 <ol><li>清洁的载玻片和污垢，胶水检查盖玻片节之间的气泡。在气泡重新安装幻灯片的情况。 </li><li>在机架上放幻灯片，并将其放置在数字扫描仪机架。在20倍放大倍率1 TMA扫描时间约6分钟。从Aperio的软件是用来选择感兴趣的领域。在软件自动对焦点选择。手动扫描区域和着力点的选择也是可能的。 </li><li>后的图像Ĥ已经产生，它可用于免疫组化染色的评价和注解。图像仍然是一个实验的结果文件，并可以向同事派发的讨论，并进一步公布的数据使用。这些图像可以被视为在Aperio一个免费软件。 </li></ol> 7。如何搜索您最喜爱的蛋白在人类蛋白质图集<ol><li>浏览到<a href="http://www.proteinatlas.org." target="_blank">www.proteinatlas.org。</a> </li><li>输入您最喜爱的蛋白质，Ensembl的ID，UniProt加入号码或HGNC名称，如胰岛素的名称。 </li><li>进入摘要页面，向下滚动到正常组织和器官的总结。概述显示在胰蛋白表达。 </li><li>点击“更多组织的数据”。你进入正常组织观看在这里你可以看到包括所有组织器官排序。 </li><li>对胰腺点击查看针对相同的蛋白质抗体的免疫组化结果。要查看一喜胰腺点击图片GH-高分辨率图像。 </li><li>有关蛋白表达在癌组织，细胞和人类蛋白质图集内的进一步验证数据的附加信息可以发现人类蛋白质图集网站浏览。 </li></ol> 8。代表结果 动画1。如何准备组织芯片的生产组织。 <a href="http://www.jove.com/files/ftp_upload/3620/3620_animation_1_Web.mov" target="_blank">点击这里查看动画1</a> 。 动画2。第TMAs。 <a href="http://www.jove.com/files/ftp_upload/3620/3620_animation_2_Web.mov" target="_blank">点击这里查看动画2</a> 。 动画3。免疫组化技术描述。 <a href="http://www.jove.com/files/ftp_upload/3620/3620_animation_3_Web.mov" target="_blank">点击这里查看动画3</a> 。<img alt="图1" src="/files/ftp_upload/3620/3620fig1.jpg" /> 图1。在人类蛋白质图集乌普萨拉进程的总体方案。 <img alt="图2" src="/files/ftp_upload/3620/3620fig2.jpg" /> 图2。捐助管芯标准化组织核心的长度标记，例如4.5毫米。 <img alt="图3" src="/files/ftp_upload/3620/3620fig3.jpg" /> 图3。手册组织微阵列。 <img alt="图4" src="/files/ftp_upload/3620/3620fig4.jpg" /> 图4。TMAs生产不同质量的例子。 （A）直和正确对齐TMA的行和列以及石蜡边缘之间有足够的空间。 （B）与一些核心公会过于接近切片时可能困难的边缘。 （C）财资市场公会已烤为TOO长在倒塌的公会不正确的模板。 <img alt="图5" src="/files/ftp_upload/3620/3620fig5.jpg" /> 图5。苏木精和曙红染色TMAs。 （A）直和对齐的列和行的部分适当削减。用坏的或脏的刀片可能会导致分裂（B）或压缩的部分（三）。 <img alt="图6" src="/files/ftp_upload/3620/3620fig6.jpg" /> 图6。抗体滴定。 （一）反应中心细胞在淋巴结显示最佳剂量的抗体，导致在一个特定的细胞质染色，无背景（棕色）。 （二）组织切片显示弱阳性。阴性或弱阳性，被发现时，初级抗体的浓度过低或如果目标蛋白是不存在的免疫染色组织。 （三）组织部分overstained由于使用初级抗体浓度过高。在D在确定由于溢出，交叉反应和假阳性的亚细胞定位的困难方舟的色彩效果。

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Histochem. , (2010).</li><li>UniProt. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic acids research. 39, D214 (2011).</li><li>Uhlen, M., Oksvold, P., Fagerberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+knowledge-based+Human+Protein+Atlas.">Towards a knowledge-based Human Protein Atlas.</a> Nature Biotechnol. 28, 1248 (2010).</li><li>Leong, A. S., Leong, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+in+immunohistology.">Standardization in immunohistology.</a> Method Mol. Cell Biol. 724, 37 (2011).</li></ol>

我们什么都没有透露。

免疫组化是迄今为止TMAs最常用的应用程序。 IHC和TMAs的组合，可以用来在几个不同的设置，如高通量筛选组织7 8蛋白的表达模式和细胞组织样本从大的病人同伙11 12及以上的基本肿瘤生物学基础9月10日 ，生物标志物的发现研究研究，包括不同的基因型/表型，分化阶段，进展和转移13。使用TMAs的一个好处是，大同伙的材料可以在同一时间进行分析，既节省了宝贵的生物材料，并确保更多的重复性实验。此外，TMA技术的使用，节省试剂成本和实验室处理时间。作为一个公会只包含数量有限的组织，组织的异质性是一个问题。根据组织类型和要解决的问题，可能需要几个样品相同specimeN。对于肿瘤组织，利用两到四个内核，每个试样建议，因为它已被证明，导致在整个组织切片13 14代表高度的准确性。根据组织组成的拳（范围从0.6毫米至2毫米）直径可以调整。组织是复杂的，既有多种不同的细胞类型，结构和细胞外基质组成。每个不同类型的可变数量的脂肪组织中含有的成分，血管，结缔组织，软骨等，会影响冲压和收集单独的内核所需的压力。因此，它是重要的前1 TMA的生产与组织定义为实验目的，练习过程中的所有步骤上，是没有任何价值的材料。 当一个公会块切片在各种不同的组织块的组成可能影响的能力，以获得高品质的部分。 CErtain组织，如皮肤，骨髓，脂肪，大脑和乳腺癌，渲染切片的TMA块困难，因为质地差异影响如何节伸展在水浴的作用，以及如何不同硬度被切断刀片等因此，建议类似的功能组组织成一个公会的脂质丰富的组织，例如，当适用。癌组织中的大多数在这个意义上是均匀的，这有利于癌症TMAs切片。处理异构类型的组织1 TMA时，可以采用替代切片稳定的芯片内核的方法。胶带的使用可以帮助，以避免在切片TMA的核心和下降的损失。胶带的使用，应采用谨慎，因为磁带可以影响切片内核的厚度和随后也IHC染色15的结果。在人类蛋白质图集设置了9x8公会的模板（不含标志）内核ARE中。它是获得最大数量的部分，并保持整个街区为代表的所有组织的重要性。为了节省试剂和扫描时间，2节被放置在一个玻璃允许9x8的最大模板的幻灯片。 主要为蛋白质信息库，通用的蛋白质资源的16包括约20.300审查人类蛋白质项，其中只有约66％，在蛋白质水平的证据。也为广大的基因，这些基因的蛋白质水平上存在的证据，有很少或没有表达的蛋白质的功能和分布的信息。对于未来的一个重要挑战是分析的分布格局和这些未知的蛋白质在人体组织的各类相对丰度。如Uniprot，ENSEMBL公布的数据显示，从医学数据库中的信息可用于为指导，以建立，如果使用抗体免疫组化染色模式病房未知的蛋白质代表实际的预定目标蛋白的蛋白质图谱。此外，其他几个验证战略的需要，以确保抗体的功能，例如蛋白质阵列的抗体，免疫印迹，免疫荧光，免疫沉淀和下拉实验功能。也许抗体的功能验证的最重要的工具是使用配对的抗体，即反对独立，互不重叠的表位的两种不同的抗体，对同一目标蛋白，比较具体分析的结果17。 的基础上编制的信息，抗体测试和滴定根据免疫组化的标准和35.000抗体在人类蛋白质图集项目的测试和验证的经验。对于超过20％的基因与蛋白质表达谱，配对抗体（2个或更多的对非重叠的表位相同的蛋白质的抗体）的数据已被用来确定最佳估计相应的蛋白表达模式的队友。配对抗体提供验证基于特定的免疫组化蛋白表达模式的最终战略。这一战略是有价值的，因为多种不同的组织包括在基本筛选增加了交叉反应的风险。还应当指出的是，某些组织一般更容易显示背景染色，如巨噬细胞，平滑肌，肾远端小管和肝细胞。额外要考虑的问题包括组织处理技术的变化，随着时间的推移，从档案材料中所采取的组织，这可能会导致假阴性或不适当的免疫组化染色阳性模式。这种现象也遇到了大断面分析。正面和负面的控制是至关重要的，应尽可能使用。为更深入的研究，包括功能分析，被迫表达的细胞株和相应的TR的具体敲anscripts（RNA干扰技术），可用于创建控件。免疫组化的最佳效果，它是重要的抗原检索系统进行测试和使用，因为福尔马林固定引起不同的化学修饰，如交联，水解希夫基地掩盖抗原18。 总之，采用TMA技术和IHC抗体为基础的蛋白质组学是一个强大的战略产生了大规模的蛋白表达数据。人体蛋白质的Atlas项目已成立，创造了人类在正常的人体组织和​​癌蛋白表达的地图。这个项目的目的是提出一个全面的人类蛋白质图集“的第一稿，到2015年。除了在正常组织和器官的蛋白质图谱，这项工作还提供了一个生物医学研究项目，包括临床生物标志物的发现工作的出发点。最终目标是在人类基因组序列的顶部添加下一个信息层，无线网络将是至关重要的一个潜在的各种疾病状态的生物学更深入的了解，并为开发新的诊断和治疗手段提供依据。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>洗缓冲区</td><td>赛默飞世尔科技</td><td> TA-999 - TT </td></tr><tr><td>柠檬酸缓冲PH6 </td><td>赛默飞世尔科技</td><td> TA-250-Pm1X </td></tr><tr><td>抗体稀释液</td><td>赛默飞世尔科技</td><td> TA-125-UD的</td></tr><tr><td> UltraVision唱片HRP的聚合物</td><td>赛默飞世尔科技</td><td> TL-125-红莲</td></tr><tr><td>民建联加上基板系统</td><td>赛默飞世尔科技</td><td> TA-125-的HDX </td></tr><tr><td>超V型块</td><td>赛默飞世尔科技</td><td> TA-125-瑞银</td></tr><tr><td>初级抗体增强</td><td>赛默飞世尔科技</td><td>的TL-125-PB </td></tr><tr><td>迈耶斯苏木</td><td> histolAB </td><td> 01820 </td></tr><tr><td> PERTEX </td><td> histolab </td><td> 00871.0500 </td></tr><tr><td colspan="3"></td></tr><tr><td> 设备名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>手册组织微arrayer </td><td> estigen，彻仪器</td><td> MTA的1 </td></tr><tr><td>瀑布切片</td><td>赛默飞世尔科技</td><td> MICROM航模355S </td></tr><tr><td>自动图像扫描仪</td><td> aperio技术</td><td> XT的</td></tr><tr><td>幻灯片自动染色系统</td><td>赛默飞世尔科技</td><td> Autostainer自动480S-2D </td></tr><tr><td>自动幻灯片为deparafinization和脱水染色系统</td><td>徕卡生物系统公司</td><td> Autostainer自动加大</td></tr><tr><td>自动玻璃coverslipper的</td><td>徕卡的BIOSystems </td><td> CV5030 </td></tr><tr><td> decloaking室</td><td>保科医疗</td><td> DC2008INTEL </td></tr></tbody></table>

production of tissue microarrays, immunohistochemistry staining and digitalization within the human protein atlas

组织芯片（TMA）技术提供了多个组织和细胞的高通量分析手段。这项技术被用于人类蛋白质图集项目内，在正常的人体组织，癌症和细胞系的蛋白质表达模式的全球分析。在这里，我们提出了1毫米的核心组件，检索，从微观选择的代表组织成一个单一的收件者公会块。在TMA块核心的数量和大小可以改变数百个0.6毫米的核心大约四十2毫米的核心。使用TMA技术的优点是，可以迅速获得大量的数据，使用单一染色协议，以避免实验变异。重要的是，数量有限，只有稀缺的组织是必要的，允许对大量病人的分析同伙1 2。约250名（4微米厚）的连续部分可以从TMA块切割，用于免疫组化staini吴以确定特定的蛋白质表达模式为250种不同的抗体。在人类蛋白质图集项目，对所有人类蛋白质产生抗体，和来自216个不同的病人，代表人类癌症的20个最常见的形式，获得相应的蛋白质图谱，从144个人和癌组织，在正常的人体组织。 TMA的免疫组化染色玻片上部分扫描创建高分辨率的图像，从病理学家可以解释和注释免疫组化的结果。图像与相应的病理基础的注释数据为研究社会公开可通过人类蛋白质图集门户网站（ <a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a> ）（ 图1）3 4。人体蛋白质图谱，提供了地图显示的蛋白质在人体中的分布和相对丰度。当前版本的锡安包含超过11万与12.238独特的蛋白，相应的超过61％的人类基因组编码的所有蛋白质的蛋白表达数据的图像。

Watch this Scientific Journal Video about Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas at JoVE.com

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

组织芯片允许一个有效的方法，从众多的组织中获得的并发信息。组织的代表零件组装成一个单一的石蜡块。从块中的部分用于免疫组化和蛋白表达模式的分析。数字扫描生成相应的图像数据分布。

组织芯片，​​免疫组织化学染色和数字化生产在人类蛋白质图集

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

production-tissue-microarrays-immunohistochemistry-staining

Research

JoVE Journal

Biology

55.7K 次观看.  Uppsala University. 组织芯片允许一个有效的方法，从众多的组织中获得的并发信息。组织的代表零件组装成一个单一的石蜡块。从块中的部分用于免疫组化和蛋白表达模式的分析。数字扫描生成相应的图像数据分布。

视频: Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

三维白色脂肪组织结构的全装染色可视化

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Y-27632 丰富成人皮肤组织中人类黑色素细胞的产量

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

牛卵巢皮质组织培养

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

丁香假单胞菌基因在植物组织中表达的单细胞分析

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

组织芯片，​​免疫组织化学染色和数字化生产在人类蛋白质图集

组织芯片，免疫组织化学染色和数字化生产在人类蛋白质图集