在作者的实验室工作是由普通医学科学研究所（GM41628）的赠款和赠款的斯托尔斯医学研究所的海伦·纳尔逊医学研究基金，在大堪萨斯城社区基金会的支持。

1代和HEK293稳定细胞系表达全长或者标记的突变版本文化抗原标记INO80或其他INO80复杂的亚基<ol><li>克隆的cDNA编码全长或突变的人INO80 ATP酶或另一INO80亚基入哺乳动物表达载体pcDNA5 / FRT用在帧中，N末端FLAG表位标签。 </li><li>出发前请确认所插入的cDNA通过DNA测序的序列。 </li><li>执行转染，成长的Flp-在HEK293细胞中，10厘米的组织培养皿在含有DMEM（Dulbecco改良的Eagle培养基），5％的谷氨酰胺的培养基中，加入10％FBS（胎牛血清）。 </li><li>当细胞达到〜70％汇合，添加到每个组织培养皿中的40微升FuGENE6转染试剂，0.5微克适当pcDNA5 / FRT表达质粒，和9.5微克pOG44，其编码的Flp重组酶的混合物中，在总体积为为800μl。 </li><li> 48小时后，trypsinize和sPLIT细胞按1:30成10 cm的培养皿的比例，并用5％谷氨酰胺，10％FBS的生长他们的​​DMEM中，并在3 100微克/毫升潮霉素B - 4周。改变时，它开始变黄的培养基（通常每3 - 5天）。 </li><li>要识别表达FLAG标签蛋白的最高水平阳性克隆，选择单独的潮霉素B抗性菌落，并将它们转移到24孔板的单井。 <ol><li>一旦细胞达到80％汇合时，收获细胞，从每孔中〜1ml的PBS和沉淀，离心1,000 xg离心5分钟。 </li><li>除去上清液后，重悬在60微升SDS-PAGE样品缓冲液将细胞沉淀。 </li><li>重悬的细胞，以SDS-PAGE和Western印迹监测国旗表达主题一半标记诱饵蛋白;保存的另一半用于将来的分析。 </li></ol></li><li>如果没有FLAG标签的人INO80蛋白在细胞裂解物检测，扩大初始克隆细胞珀普通过电镀从单个孔24孔板中的细胞分成15厘米的组织培养皿中进一步分页。 <ol><li>到15 cm的培养皿，接近融合的细胞重悬在冰冷的PBS中，并转移到50ml锥形管中收获细胞。 </li><li>通过添加额外的PBS带来的50 ml的终体积。 </li><li>沉淀的细胞在1,000×g离心5分钟，并除去尽可能多的上清液。 </li><li>重悬在1ml的Lys450缓冲细胞沉淀（20mM的HEPES-氢氧化钠pH值为7.9，450 mM氯化钠，0.5％的TritonX-100，10mM的氯化钾，4毫米MgCl 2，0.2毫摩尔EDTA，10％甘油，1mM DTT， 200μM的PMSF，和1:1,000蛋白酶抑制剂混合物）。 注：这里和其他地方，立即开始实验前，随时添加DTT，PMSF和蛋白酶抑制剂来缓冲。 </li><li>使用20微升抗FLAG琼脂糖凝胶免疫沉淀FLAG标签从所​​得的全细胞裂解液的蛋白质，并分析FLAG洗脱物用免疫蛋白印迹。 [为了与免疫详情oprecipitation步骤，请参阅步骤5] </li></ol></li><li>制备所需的克隆细胞系12的冷冻储存，并将它们存储在液氮中直至使用。 </li></ol>在滚瓶2。成长的HEK293细胞系对于大规模制备INO80复合物，培养的细胞在10 - 20滚瓶;一个典型的产量从每滚瓶〜1毫升包装细胞。 <ol><li>到每个滚瓶中，加入200毫升的DMEM，5％的谷氨酸，和10％小牛血清，无潮霉素B. </li><li>从单个近汇合15cm培养皿转移所有的细胞到每个滚瓶</li><li>地方滚瓶到37℃，转瓶培养器和旋转0.2转。 </li><li>一旦细胞达到〜70％汇合，倒出并丢弃的媒介。 </li><li>加入约50毫升冰水中冷PBS至各瓶中。拿着瓶水平，轻轻地旋转松开细胞单层。 </li><li>传输重悬细胞250ml的PLASTIC锥形瓶置于冰上。 </li><li>冲洗滚瓶额外的PBS，依次传递从瓶到瓶。当冲洗液不再清晰（通常被用于漂洗约5瓶后），将其添加到细胞悬浮液。 </li><li>离心沉淀细胞，在400×g离心10分钟。 </li><li>轻轻悬浮细胞在PBS，将它们组合成一个单一的250毫升锥形瓶中，并保持在冰上，直到进一步的处理。 </li></ol> INO80亚基细胞表达的另一个FLAG标签INO80亚基3的siRNA介导的击倒为了获得缺少一个亚基INO80 subcomplexes，使用标志，免疫纯化，净化的siRNA处理的细胞或细胞中稳定表达shRNA的INO80复合物。 “反向”的siRNA（小干扰RNA）转染这里描述的协议被优化为HEK293细胞15 cm的培养皿生长。该协议是在细胞和SHO的一个15cm培养皿ULD扩大规模相应地取决于所需的细胞数。要准备从siRNA治疗的细胞INO80复杂的生化有用的数额，应扩大到40 15 cm的培养皿生长的培养;这将产生约2 - 4毫升填充细胞沉淀。 <ol><li>生长的HEK293细胞稳定表达INO80复合物的所希望的亚基附近汇合15 cm的培养皿。 </li><li>添加的siRNA的再悬浮缓冲液的量（20毫米氯化钾，6毫米肝素钠，pH值7.5，和0.2毫米氯化镁2）充分准备的siRNA的50微米原液含有冻干的siRNA管。吸取溶液，上下几次，并培育一个nutator 30分钟，在室温，确保siRNA的完全溶解。 </li><li>制备含siRNA和转染试剂转染的鸡尾酒。混合10微升的50μM的siRNA的储备溶液与32微升的Lipofectamine RNAiMAX，并将其添加到4ml的Opti-MEM的低血清培养基用非常温和混合;人低所有试剂在使用前平衡至室温。 </li><li>在室温下孵育该混合物30分钟。 </li><li>制备的Flp-在HEK293细胞中稳定表达所需INO80亚基转染。 <ol><li>在步骤3.4的孵化，在15 cm的培养皿一次，用RT PBS洗涤细胞。 </li><li>除去PBS后，把细胞用1ml胰蛋白酶只是直到他们开始抬离板。 </li><li>立即加入10 ml完全培养基（DMEM + 5％谷氨酰胺+ 10％FBS）与胰酶消化细胞，轻轻混匀。通过离心收集的细胞在1,000×g离心5分钟，在室温。 </li><li>重悬在〜4毫升完全培养基将细胞沉淀，并用血球计计数细胞再悬浮。 </li><li>稀释细胞完全培养基中至〜5.4×10 6个/ ml的浓度。 </li></ol></li><li>向每个15cm培养皿，加入15 m​​l完全培养基然后加入4ml染鸡尾酒。轻轻摇动，以确保中期和转染鸡尾酒是thoroughly混合。最后，添加一个ml的细胞悬液，再轻轻摇晃均匀分散细胞。 </li><li>在37℃，5％CO2 培养箱 60小时培养后，轻轻取出介质，悬浮细胞在冰冷的PBS，并立即着手准备核提取物。 </li></ol> 4，制备核提取物的这个过程已经被修改狄格南13的协议，并且可以放大或缩小根据起始细胞沉淀物的大小。典型地，将1ml填充细胞沉淀收率1最终核提取毫升。所有的缓冲区应该是冰冷的，而所有步骤应在冷室或冰面上进行，如果合适的冷室中不可用。 <ol><li>隔离核： <ol><li>轻轻地在4℃下的细胞转移至合适大小的（15或50毫升）刻度锥形试管和自旋在1,000 xg离心10分钟。 </li><li>除去上清液，并测量红细胞象素的体积让。 1毫升包装细胞对应于〜3×10 8个HEK293细胞。 </li><li>添加5包装细胞的缓冲液A的体积（10mM的HEPES，pH值7.9，1.5毫米MgCl 2，10 mM的氯化钾和新加入1毫摩尔DTT，200μMPMSF，和1:1,000蛋白酶抑制剂混合物）。重悬细胞沉淀轻柔吹打。 </li><li>在冰上孵育恰好10分钟。 </li><li>沉淀细胞在1,000×g离心10分钟，在4℃下，并除去上清液。 </li><li>将细胞沉淀物的大小应增加达2倍的孵育在缓冲液A中</li><li>将细胞重悬于缓冲液A中的两个压缩细胞体积，细胞悬浮液转移到一个适当大小的Dounce组织匀浆器。如果少于2毫升包装细胞开始，使用7毫升均质; 2 - 4毫升包装细胞，用15毫升均质;并为10个或更多毫升装电池，使用40毫升均质。 </li><li>均质的细胞悬液用Dounce匀浆联合国松散玻璃杵直到90％的细胞用1％的台盼蓝染色为阳性。 </li><li>的悬浮液转移到一个45毫升高速离心管中并旋转以25,000 xg离心20分钟，在4℃下，在JA-17或类似的转子（ 见表的材料/设备 ）。 </li><li>从该核沉淀，除去上清液，其含有细胞质蛋白或分馏过程中泄漏出来的核蛋白质。 </li></ol></li><li>细胞核中提取盐： <ol><li>加入缓冲液C（20mM的HEPES，pH值7.9，25％甘油，1.5mM的MgCl 2的，0.2mM的EDTA，和新加入1毫摩尔DTT，200μMPMSF，和1:1,000蛋白酶抑制剂混合物）的核沉淀;使用2.5毫升缓冲液C为每3毫升开始收集细胞体积（〜1×10 9个细胞）。 </li><li>用玻璃棒或吸管，逐出该管的壁的核颗粒和整个混合物转移到一个适当大小的Dounce匀浆。 </li><li>通过均质个悬浮核Ë混合搭配宽松杵两招。 </li><li>转移悬浮细胞核部分进入冷藏烧杯中。选择的烧杯中，使得悬浮液将填充该烧杯，以至少0.5厘米深。 </li><li>以提取从染色质或其他不溶性结构的核蛋白质，该悬浮液的盐浓度逐渐增加至0.42 M氯化钠，滴加5M的NaCl，同时轻轻地搅拌该悬浮液在冰上吸管或玻璃棒;一旦所有的5M的NaCl的已被添加，则溶液变得非常粘稠的或凝胶状。计算的5M NaCl的需要，根据下面的公式，使该溶液的0.42 M氯化钠的最终浓度的体积： <img alt="式（1）" fo:content-width="4.5in" src="/files/ftp_upload/51720/51720equation1.jpg" width="500" /></li><li>粘性悬浮小心转移到10ml聚碳酸酯管的类型70.1钛转子（或同等学历），或70毫升聚碳酸酯瓶的45型钛Øř类似的转子（见表材料/设备）。如果用10 ml离心管或盖组件，如果用70 ml瓶装封口膜密封严密。 </li><li>慢慢地摇动密封管在4℃下用章动器30分钟。 </li><li>旋样本中的45型Ti或70.1 Ti转头30分钟12万XG在4℃。 </li><li>将上清转移到一个单一的塑料管或瓶。此上清液为核提取物，并将沉淀包含染色质和其他核碎片。 </li><li>分裂的核提取物成方便的尺寸等分，冷冻它在液氮中，并储存在-80℃。 </li></ol></li></ol>人类INO80或INO80 Subcomplexes 5。免疫亲和纯化<ol><li>解冻冻结核提取物，含手部之间的台式或卷管提取物，直到冷冻材料管的地方就变成了泥浆。然后将试管置于冰上或在冷室中，直至提取物是完全吨hawed。 </li><li>传送解冻核提取物至10ml聚碳酸酯超速离心管中，并旋以100,000 xg离心20分钟，在4℃下，在类型70.1 Ti转头或等效以去除可能在冻融循环已经形成的任何沉淀物。 </li><li>将上清转移到一个15毫升的锥形管。加入新鲜的DTT，PMSF和蛋白酶抑制剂混合物至最终浓度为1mM DTT，200μMPMSF，和1:1,000蛋白酶抑制剂混合物。 </li><li>要准备抗FLAG琼脂糖的免疫纯化，用P200或类似吸管尖端从中年底已被切断，用干净的手术刀转移200微升抗FLAG琼脂糖珠50％的淤浆到1.5 ml离心管或刀片。 </li><li>沉淀珠离心的台式微型离心机在8000 XG，持续30秒。除去上清液，并通过再悬浮珠粒在1ml的Lys450缓冲洗珠，并沉淀小珠以8,000 xg离心30秒。洗的BEADS两次以上。 </li><li>重悬在约100微升的核提取物使用的是P200或类似音量可调移液管与尖端从该端部已经被切断，用干净的手术刀或剃刀刀片，并且使用相同的尖，转印的洗涤的抗FLAG琼脂糖珠再悬浮的珠至15毫升锥形管中含有的提取物。重复几次，直到所有的球都被转移到15ml试管中。 </li><li>孵育提取/珠混合物4小时，在4℃用在实验室旋转体缓慢转动。可选：包括BENZONASE在25单位/毫升的浓度去除污染的DNA。 </li><li>收集标志琼脂糖离心1000×g离心5分钟，在4℃。 </li><li>悬浮于10ml Lys450洗，孵化5分钟，在4℃下轻轻摇动一个nutator。沉淀小珠在1,000 xg离心5分钟，在4℃下。 </li><li>悬浮在100 - 150微升Lys450和转运珠，以1.5 ml离心管中。合作ntinue冲洗的15毫升锥形管，100 - 150微升Lys450的，直到所有的珠粒已被转移到离心管中。 </li><li>在微型降速珠在8000 XG为30秒，在4℃。用1ml EB100缓冲液（10mM HEPES pH为7.9，10％甘油，100mM的氯化钠，1.5mM的MgCl 2的，0.05％的TritonX-100，和新加入1毫摩尔DTT，200μMPMSF洗三次以上用1ml Lys450和一次，和1:1,000蛋白酶抑制剂混合物）。 </li><li>为了洗脱结合的蛋白，加入含0.25毫克/毫升1X FLAG肽200微升EB100缓冲区。孵育一个nutator在4℃每次30分钟。 </li><li>在微量离心沉淀珠粒在8000×g离心30秒，在4℃下。将上清液转移，它包含洗出INO80复合物，一种新的微量离心管中。 </li><li>重复洗脱四次，并集中所有的上清液到一个单一的管。 </li><li>从洗脱的蛋白级分除去任何残留的FLAG-琼脂糖珠，通过一个空的离心柱通过洗脱液。 </li><li>浓缩洗脱的蛋白级分〜10倍使用超离心过滤装置（50,000分子量截留值）。 </li><li>要删除FLAG肽，通过两个脱盐柱依次传递浓缩的蛋白质部分。 </li><li>分纯化，脱盐的蛋白质级分入20微升的等分试样，在液氮中冷冻，并保存于-80℃。 </li><li>银染胶或免疫印迹分析INO80或INO80 subcomplexes的亚基组成，并估计它们的浓度用已知浓度的标准INO80重组亚单位准备半定量免疫印迹。 </li></ol>

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L., Chubet, R. G., Vizard, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoaffinity+purification+of+FLAG+epitope-tagged+bacterial+alkaline+phosphatase+using+a+novel+monoclonal+antibody+and+peptide+elution,+Biotechniques.">Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution, Biotechniques.</a> 16 (4), 730-735 (1994).</li><li>Barker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> At the Bench: A Laboratory Navigator. 10, 207-245 (2005).</li><li>Dignam, J. D., Lebovitz, R. M., Roeder, R. 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作者宣称，他们没有竞争的经济利益。

从高等真核生物多亚基哺乳类染色质重塑复合物的结构和功能的研究已经受到阻碍制备含有突变亚基或完全缺乏某些亚基这种配合物的生物化学有用量的难度。有许多技术障碍：第一，基因操作在哺乳动物细胞中已经在技术上具有挑战性的并且耗时的。不同于酵母细胞，其基因组可以容易地编辑和定向用重组工程技术，在哺乳动物基因组中是更复杂的结构和较少受重组工程干预。因此，缺失或哺?...

进化上保守的SNF2家庭染色质重塑复合物染色质组织和DNA可达1的主要监管机构。这些重塑复合物总是包括中央SNF2状ATP酶亚基，其中，在某些情况下，装配有各种辅助蛋白，并形成多亚基大分子装配体。研究ATP依赖的染色质重塑过程的分子细节，以理解亚基和/或结构域的结构的特定子集的贡献，以复合物的活性是很重要的。这样的分析要求，以产生缺乏特定蛋白质亚基或结构域的结构高度纯化的突变体复合物的能力。 的依赖ATP的染色质重塑复合物之前的结构-功能研究已经广泛地聚焦在酵母模型系统上，由于酵母基因组的优异的可操作性（参见，例如，文献1-4）。由于养护其中同源重塑复合物亚基组成和功能，结构和酵母重塑复合物的功能研究高等真核生物提供了重要的见解的同行。然而，明显的种属特异性重塑复合物之间的差异确实存在，从增益或物种特异性的亚基，增益或保守的亚单位的种特异性结构域的损失，和序列变异性的保守亚基的保守结构域之内的损失所导致。这样的差异可以在原则上需要高等真核细胞，以适应新的分子和细胞的环境中进行驱动。因此，理解的高等真核质重塑复合物亚基如何向核小体重构过程是有价值的，因为它不仅揭示了ATP依赖的染色质重塑过程的基本机制的光，而且还可以提供有价值的洞察机制，其中的染色质结构在集锦和基因表达她真核生物规管。 到目前为止，已经出现了只有有限的多亚基哺乳动物染色质重塑复合物的结构和功能的研究，部分是由于难以获得生化定义染色质重塑复合物及其亚。我们已经部分地规避这些困难与下面描述的方法，其中免疫亲和纯化用于制备从人体细胞完好INO80或INO80 subcomplexes稳定表达N末端​​FLAG表位标记的野生型或INO80 5-7的突变版本（ 图1） 。以获得从人体细胞完好INO80复合物，FLP介导的重组被用于产生转基因的HEK293细胞系中稳定表达FLAG表位标签的编码cDNA的INO80复合物8-10的亚单位组成。因为过度表达INO80亚基可以是有些有毒，有必要分离并保持下选择性共克隆细胞系条件，以确保在需要扩张大规模细胞培养物的多通道的稳定的转基因表达。要获得含有亚单位的一个子集较小INO80 subcomplexes，我们已经成功地使用两种方法（ 图2A，B）。在所述第一，我们生成的HEK293 FLP-在细胞系中稳定表达INO80的突变版本，缺乏具体的亚基5所需的相互作用结构域。或者，siRNA的介导击倒用于消耗从细胞中表达适当的FLAG标记的INO80亚基（未发表的数据）所需的子单元。最后，为了净化人类INO80复合物，FLAG琼脂糖基于色谱11被用于从核提取物丰富的含INO80馏分，从而有效地减少了污染的胞质蛋白在含有纯化INO80或INO80 subcomplexes的最后部分的存在。

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</table>

generation and purification of human ino80 chromatin remodeling complexes and subcomplexes

INO80染色质重塑复合物通过催化ATP依赖的核小体重塑核监管动态和DNA的可访问性。人类INO80物包括14蛋白亚基包括INO80一个SNF2般的ATP酶，它同时作为催化亚基和支架的复合物的组装。另一亚基的功能和由此来的INO80复合物的染色质重塑活性它们贡献的机制仍然知之甚少，部分原因是由于产生INO80组件在人细胞或异源表达系统的挑战。这JOVE协议描述的程序，可以让那些缺乏亚基或亚基的一个子集人类INO80染色质重塑subcomplexes的净化。 N末端FLAG表位标签的INO80基因被稳定地导入人胚胎肾（HEK）用FLP-介导的重组293细胞系。在该INO80复合物的亚基的一个子集是B中的事件ê删除，1表示是缺乏必要对这些亚基组装的平台，而不是突变INO80蛋白质。在该事件的个体亚基是被耗尽的，针对这一亚基成的HEK 293细胞系的稳定表达FLAG 1 transfects的siRNA标记INO80 ATP酶。核提取物的制备和免疫标志进行充实含INO80衍生蛋白组分。纯化INO80 subcomplexes的组合物然后可以使用的方法，例如免疫印迹，银染色法和质谱法进行分析。产生的INO80和INO80 subcomplexes根据该协议可以使用各种生化测定法，其中在附图JOVE协议描述来进一步分析。这里描述的方法可以适用于任何哺乳动物多亚基染色质重塑的结构和功能特性的研究和改进的配合物。

图1示出的流程图总结了用于产生，纯化和表征人类INO80 ATP依赖的染色质重塑复合物的过程。 如示于图2和3中 ，这些程序使缺乏各种亚基，从而使这些缺失亚基INO80的酶活性的贡献后续的生化分析野生型INO80和INO80 subcomplexes的产生， 图2描述了两种策略我们用于定义INO80复合物的结构和生成INO80 subcomplexes。在第一阶段， ?...

Watch this Scientific Journal Video about Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes at JoVE.com

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

这个协议描述了用于产生和纯化野生型和人INO80染色质重构复合物的突变版本中的一个过程。表位标记的INO80亚基的版本都稳定地表达在HEK293细胞中，并完全配合物和配合物缺乏亚基的特定集合是通过免疫亲和层析纯化。

产生和人类INO80染色质重塑复合物及其亚纯化

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

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9.7K 次观看.  Stowers Institute for Medical Research. 这个协议描述了用于产生和纯化野生型和人INO80染色质重构复合物的突变版本中的一个过程。表位标记的INO80亚基的版本都稳定地表达在HEK293细胞中，并完全配合物和配合物缺乏亚基的特定集合是通过免疫亲和层析纯化。 

视频: Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

A型流感病毒的核蛋白复合物的分离纯化及可视化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

科研

生物学

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

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 ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

制备，纯化和磁共振成像造影剂的使用稀土配合物的表征

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

侵入细胞DISC1蛋白种类的产生，纯化及表征

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

生化分析的分析ATP依赖的染色质重塑酶活性的影响

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...