Name: generation and purification of human ino80 chromatin remodeling complexes and subcomplexes
Uploaded: 2014-10-23T13:00:00.000+00:00
Duration: 8 min 44 s
Description: Watch this Scientific Journal Video about Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes at JoVE.com

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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+transcription+initiation+by+RNA+polymerase+II+in+a+soluble+extract+from+isolated+mammalian+cell+nuclei.">Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian cell nuclei.</a> Nucleic. Acids. Res. 11 (5), 1475-1489 (1983).</li></ol>

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，缺失或哺乳...

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。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>Glutamax-I (stablized glutamine)</td><td>Life Technologies</td><td>35050-079</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>SAFC</td><td>12176C</td><td></td></tr><tr><td>FuGENE6 transfection reagent</td><td>Promega</td><td>E2312</td><td></td></tr><tr><td>Hygromycin B, sterile in PBS</td><td>AG Scientific</td><td>H-1012-PBS</td><td></td></tr><tr><td>pcDNA5/FRT vector</td><td>Life Technologies</td><td>V6010-20</td><td></td></tr><tr><td>Flp-In HEK293 cells</td><td>Life Technologies</td><td>R780-07</td><td></td></tr><tr><td>pOG44 Flp-Recombinase Expression Vector</td><td>Life Technologies</td><td>V600520</td><td></td></tr><tr><td>EZview Red ANTI-FLAG M2 Affinity Gel</td><td>Sigma</td><td>F2426</td><td></td></tr><tr><td>calf serum</td><td>SAFC</td><td>12138C</td><td></td></tr><tr><td>TARGETplus SMARTsiRNA pool</td><td>Dharmacon / Thermo Scientific</td><td>various</td><td></td></tr><tr><td>5x siRNA resuspension buffer</td><td>Dharmacon / Thermo Scientific</td><td>#B-002000-UB-100</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Life Technologies</td><td>13778</td><td></td></tr><tr><td>Opti-MEM Reduced Serum Medium</td><td>Life Technologies</td><td>51985-091</td><td></td></tr><tr><td>PBS</td><td>Cellgro</td><td>45000</td><td>VWR</td></tr><tr><td>TrypLE (trypsin)</td><td>Life Technologies</td><td>12604</td><td></td></tr><tr><td>1x FLAG Peptide</td><td>Sigma</td><td>F3290</td><td></td></tr><tr><td>Micro Bio-Spin Chromatography Column</td><td>Bio-Rad</td><td>737-5021</td><td></td></tr><tr><td>Amicon Ultra Centrifugal Filter Device (50k MWCO)</td><td>Amicon</td><td>UFC805024</td><td>Fisher Scientific</td></tr><tr><td>Zeba Desalting Columns</td><td>Thermo Scientific</td><td>89882</td><td></td></tr><tr><td>Anti-FLAG M2 antibody, mouse</td><td>Sigma</td><td>F3165</td><td></td></tr><tr><td>Anti-FLAG M2 antibody, rabbit</td><td>Sigma</td><td>F7425</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma</td><td>P8340</td><td></td></tr><tr><td>benzonase</td><td>Novagen</td><td>70664</td><td></td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>JS-4.2 rotor in a J6 centrifuge</td><td>Beckman-Coulter</td><td>339080</td><td></td></tr><tr><td>JA-17 rotor</td><td>Beckman-Coulter</td><td>369691</td><td></td></tr><tr><td>10 ml polycarbonate tubes</td><td>Beckman-Coulter</td><td>355630</td><td></td></tr><tr><td>70 ml polycarbonate bottles</td><td>Beckman-Coulter</td><td>355655</td><td></td></tr><tr><td>Type 45 Ti rotor</td><td>Beckman-Coulter</td><td>339160</td><td></td></tr><tr><td>Type 70.1 Ti rotor</td><td>Beckman-Coulter</td><td>342184</td><td></td></tr><tr><td>BD Clay Adams Nutator Mixer</td><td>BD Diagnostics</td><td>15172-203</td><td>VWR</td></tr><tr><td>Glas-Col Tube/Vial Rotator</td><td>Glas-Col</td><td>099A RD4512</td><td></td></tr><tr><td>PCR thermal cycler PTC 200</td><td>MJ Research</td><td>PTC 200</td><td></td></tr><tr><td>roller bottle incubator</td><td>Bellco biotechnology</td><td>353348</td><td></td></tr><tr><td>Immobilon-FL Transfer Membrane 7 x 8.4</td><td>Millipore</td><td>IPFL07810</td><td></td></tr><tr><td>lubricated 1.5 ml microcentrifuge tubes</td><td>Costar</td><td>3207</td><td></td></tr></tbody></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 ...

generation-purification-human-ino80-chromatin-remodeling-complexes

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

DNA and Chromosome Structure

SCIENTISTS IN ACTION

9.8K 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

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

科研

遗传学

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

小鼠棕色脂肪组织的染色质免疫沉淀

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

核糖体组蛋白的体外泛化和二次基质测定

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

生物化学

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

使用色素免疫沉淀-外核酶（ChIP-Exo）对小鼠干细胞衍生神经元中的蛋白质-DNA相互作用进行高分辨率映射

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

生物学

从酵母细胞中分离基因座特异性染色质

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

作者聚焦：使用高富集位点特异性染色质分离方案推进染色质研究并克服限制 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

酿酒酵母的单拷贝基因位点染色质纯化

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

生物质的使用限制金额高效的染色质免疫沉淀

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

产生和人类INO80染色质重塑复合物及其亚纯化

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使用色素免疫沉淀-外核酶（ChIP-Exo）对小鼠干细胞衍生神经元中的蛋白质-DNA相互作用进行高分辨率映射

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

酿酒酵母的单拷贝基因位点染色质纯化

酿酒酵母的单拷贝基因位点染色质纯化

酿酒酵母的单拷贝基因位点染色质纯化

生物质的使用限制金额高效的染色质免疫沉淀

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景