这项工作是由美国国立卫生研究院资助CA141009和GM39067支持。我们感谢E.帕内尔和R. Yarrington的的笔试部分的意见。

1。分离小鼠脾幼稚的CD4 T细胞<ol><li>牺牲了鼠标，以人道的方式，符合机构的动物护理和使用委员会（IACUC）协议。解剖脾，并将其放置在培养皿中含有10毫升含10％FBS的DMEM。 </li><li>粉碎使用磨砂两端的两个载玻片释放脾脾。细胞悬液转移15毫升锥形管中。 </li><li>通过离心收集细胞，在200×g离心（〜1,200 rpm下一个典型的转子直径的临床离心机）5分钟，在4℃下</li><li>将细胞重新悬浮在2ml ACK缓冲液裂解红血细胞，在室温（RT）1分钟。加入8毫升与胎牛血清（FBS）的DMEM终止反应。 </li><li>收集细胞以200×g离心5分钟，在4℃下</li><li>在5毫升含FBS重悬细胞和通过70微米网状过滤器（BD猎鹰，猫＃352350）。计数细胞，并继续孤立幼稚的CD4 T细胞CD4细胞分离试剂盒（美天旎，猫＃130-095-248）使用制造商的说明使用。收藏幼稚的CD4 + T细胞通过离心分离，在200×g离心5分钟，在4℃下在10毫升与胎牛血清（FBS）的DMEM培养液中重悬细胞。 </li></ol> 2。染色质的制备<ol><li>至终浓度为1％的DMEM将细胞悬浮液加入37％的甲醛，并轻轻摇动15分钟，交联在RT（ 例如使用Nutator），DNA:蛋白质复合物。 </li><li>停止，加入1 M甘氨酸至终浓度为125 mM的交联。继续摇动5分钟，在室温。 </li><li>收集细胞以200×g离心5分钟，在4℃下</li><li>洗细胞，重新悬浮于5毫升冰冷的PBS含蛋白酶抑制剂。在冰冷的PBS含蛋白酶抑制剂，共3次洗涤，并通过离心收集细胞，在4℃下</li><li>重悬在1ml的细胞冰冷的细胞裂解液含有蛋白酶抑制剂。在冰上孵育15分钟。可以由一小等分的细胞重新悬浮在0.4％台盼蓝溶液（Sigma公司，年龄组＃T8154），用显微镜观察细胞裂解的效率。 </li><li>通过离心收集细胞核在200 XG（一般为〜1200转）5分钟，在4℃下小心弃上清。 </li><li>重悬在500μl的核含蛋白酶抑制剂的裂解缓冲液中的原子核。在冰上孵育15分钟。 </li><li>超声细胞，使用一个Misonix的超声波清洗3,000，探头尺寸1.6毫米:输出电平，15秒连拍，在冰上的4倍。每个样品必须在1-2分钟，在冰上冷却，然后再次超声处理样品，以防止过热。过热会引起交叉链接的逆转。 </li><li>超声处理的染色质在16000×g离心（〜13,200 rpm，以微量离心）离心5分钟，在4℃下</li><li>取20微升等分明确supernatant，加DNA上样染料，并检查通过超声处理的DNA通过电泳在2％琼脂糖凝胶（对于大多数应用超声处理的DNA的理想大小为200-500碱基对）。 </li><li>确定DNA的浓度用紫外分光光度计。设立染色质免疫沉淀反应，剪切染色质可以立即使用或储存于-80℃。通常情况下，我们取得2-3×10 6纯化的CD4 T细胞每鼠7.5-10微克DNA。 </li></ol> 3。染色质免疫沉淀（所有步骤必须在0-4°C） <ol><li>超声处理的染色质稀释的DNA浓度为5-10微克/毫升（总体积= 1毫升）中的ChIP与蛋白酶抑制剂的缓冲液稀释。 </li><li>减加入100μl（10％）作为输入。存放在冰上。 </li><li>分装450微升成两个1.7毫升离心管型对照（鼠或兔IgG）和抗体的标记。如果有多个相同的同种型的抗体的使用，一单一的同种型对照醇将足以进行这种分析。您将需要多个同型对照，如果使用不同的动物源或同型抗体。 </li><li>添加2-5微克的特异性抗体，这取决于所使用的抗体的特异性，或同种型对照的各管。 </li><li>摇动使用Nutator管在4℃下过夜，以允许染色质抗体复合物的形成。 </li><li>向上述混合物中加入25μl的G蛋白磁珠（1:1淤浆悬浮有孔玻璃珠），并允许岩石至少2小时，在4℃。从我们的供应商的有孔玻璃珠，可以直接使用。 </li><li>离心管放置磁性的立场上，让珠收集磁化侧。 </li><li>小心删除解决方案的愿望而不扰乱珠。 </li><li>加入低盐洗涤液1毫升，，并允许轻轻摇动5分钟一个nutator。收集珠使用磁性的立场和取出洗涤液。重复一次。</li><li>高盐洗涤溶液中加入1毫升，并允许为5一个nutator分钟摇滚。收集珠使用磁性的立场和取出洗涤液。重复一次。 </li><li>氯化锂洗涤溶液中加入1毫升，并允许为5一个nutator分钟摇滚。收集珠使用磁性的立场和取出洗涤液。重复一次。 LiCl的使用提高与有孔玻璃珠的非特异性的染色质相互作用的有效去除。 </li><li>加入1毫升TE的解决方案，并允许为5一个nutator分钟摇滚。收集珠使用磁性的立场和取出洗涤液。 </li><li>从有孔玻璃珠，加入250微升的洗脱缓冲液洗脱DNA。在室温下摇动15分钟。吸出的洗脱液保存这种材料一个新的1.7 ml离心管。再重复一次，并结合双方的洗脱。丢弃的珠子。 </li><li>为了扭转交叉链接添加氯化钠5米至终浓度为0.3米和1微升的核糖核酸酶A（20米<html>克/毫升）的洗脱下来的DNA。在步骤3.2中，保存到输入端，使该卷500微升的洗脱缓冲液中加入400μl。到输入端添加氯化钠0.3 M，1微升的核糖核酸酶A，10微升的0.5M EDTA，20微升的1M的Tris-HCl pH 6.5的蛋白酶K（20毫克/毫升）和1μl。 </li><li>孵育过夜管在65℃下，在干燥的加热块。为了避免蒸发的样品，密封管上放置一个重量，让他们打开。也可用于杂交炉。 </li><li>让冷却至RT管。加入1毫升100％乙醇中，孵育2小时过夜，在-80℃下沉淀DNA。 </li><li>离心管，以16,000×g 15分钟沉淀的DNA。洗涤DNA沉淀用70％乙醇和空气干燥的颗粒一次。 DNA沉淀重悬在100μl的高压灭菌蒸馏水。 </li><li>纯化DNA用QIAquick离心柱，在总体积为50μl的洗脱缓冲液洗脱。这DNA是准备用于PCR。 </li></ol>"&gt; 4。制备PCR反应（所有步骤必须进行冰） <ol><li>收集所有的芯片和相应的输入样本的DNA样本。此外，收集所有的PCR试剂和引物进行有针对性的区域，以及一个控制区。使用"热启动"Taq DNA聚合酶。在此过程中，我们将使用的SYBR Green为基础的检测DNA扩增量化，如果有必要，可以用来但在盒子定量PCR试剂盒。 </li><li>进行连续稀释的DNA模板以生成标准曲线进行定量PCR分析:例如，在洗脱缓冲液中的10％，1％，0.1％和0.01％（在步骤3.18中的QIAquick离心柱）。的浓度范围，在此标准曲线模板集增量的ChIP富集的基础上预期的水平而有所不同，分配输入10μl的DNA样本的96 - 孔板的各孔中，猫＃T-3182-1（Genemate ），一式两份。 </li><li>免除10毫升芯片型对照DNA以及在各自的特异性抗体井，一式三份。 </li><li> "大师"包含任一目标的引物或控制引物（引物各0.1微米终浓度）混合和分配到各井的10微升。例如，在下面的模板免除主混合井含有针对性的引物标记为绿色和红色标记，分配主控制引物组合成井。 </li><li>盖上的PCR板与光学塑料密封膜和离心500 XG在临床离心机（1200转），1分钟RT收集一切在井底。 </li><li>启动PCR反应。我们将使用的LightCycler 480 II（罗氏公司）经营的LightCycler 480SW 1.5软件运行在这个例子中的qPCR。 </li></ol>预孵育:1周期:95℃/ 5分钟。 放大:40-45周期:94°C / 5秒，60°C / 5秒，72°C/10秒（退火温度应为2-3°C小于熔化TEMPERATUR辰引物）。 熔解曲线:1个周期。 冷却方式:1个周期。 <table border="1"><tbody><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 11 </td><td> 12 </td></tr><tr><td>一</td><td> 10％输入</td><td> 10％输入</td><td>抗体亚型</td><td>具体</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>乙</td><td> 1％的输入</td><td> 1％的输入</td><td>抗体亚型</td><td>具体</td><td></td><td></td><td></td><td></td><td></td><td></td&gt; <td></td><td></td></tr><tr><td> Ç </td><td> 0.1％的输入</td><td> 0.1％的输入</td><td>抗体亚型</td><td>具体</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> ð </td><td> 0.01％输入</td><td> 0.01％输入</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> ë键</td><td> 10％输入</td><td> 10％输入</td><td>抗体亚型</td><td>具体</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> F </td><td> 1％的输入</td><td> 1％的输入</td><td>抗体亚型</td><td>具体</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> ĝ </td><td> 0.1％的输入</td><td> 0.1％的输入</td><td>抗体亚型</td><td>具体</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> ħ </td><td> 0.01％输入</td><td> 0.01％输入</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>绿色环保:使用针对性的区域引物。 红色:使用控制区域的引物。 5。分析<ol><li>计划的qPCR软件定量DNA的绝对数量。重要的是，使用的标准曲线内插未知的具体的和同种型的样品中的DNA量允许在样本中的浓度的范围内设置的所有引物的质量和效率的评价和匹配。此外，它也提供了铁道部E可靠方法，CQ（C T C P）值转换到最终相对倍浓缩的结果比usingΔΔCq和相关方法。 </li><li>进入采样编辑器和包含输入样本的各种稀释液（10％，1％，0.1％和0.01％）与标准曲线值的井马克。同时要注明井未知的芯片样品，PCR板奠定了完全一样。在使用相同的软件在其他的qPCR机，指定子集对应每对引物，标准曲线值和实验型对照样品相应的反应。 </li><li>在LightCycler软件，去子集编辑器和标记的子集，包括井与输入样本以及未知的芯片样品。未知样品的定量使用产生的，用于分析输入样本的已知浓度的标准曲线。在使用相同的软件在其他的qPCR机，候子集的对应元素onding每个引物对所有的反应。 </li><li> PCR扩增完成后，"ABS Quant/2nd衍生最大"进行分析和选择的子集进行分析，并按下OK。在使用相同的软件在其他的qPCR机，CQ值转换的DNA量在每个子集。 </li><li>按"计算"，这将产生使用输入样本的已知浓度的标准曲线，将显示未知样品中存在的DNA的绝对量。 </li><li>如果剪切的DNA的质量是好的，并且，如果引物特异性结合的对象区域，PCR扩增效率应该是接近2。 </li><li>执行"以旧换新调用"相同的子集，如果有一个熔解曲线分析。单峰表示，只有一个特定的产品扩增。随着电泳PCR产物通过琼脂糖凝胶中的DNA梯状条带，扩增特定的DNA也可以进行测试。 </li><li>将数据导出为一个标签带分隔符的文本文件，它可以在Microsoft Excel中打开，作进一步的分析。 </li><li>使用Microsoft Excel中，划分的DNA量进行有针对性的引物特异性抗体同型对照。重复此步骤，为控制区引物。如果有多个相同的同种型的抗体被用于不同的免疫沉淀，标准化每个这些值与从相同的同种型对照。此外，如果使用的多个对象的引物对，DNA的数量从一个单一的控制引物对集应用于每个目标的归一化（ 图1和2）。的输出值表示在每个位置相对于非特异性抗体背景免疫沉淀的特异性抗体的免疫沉淀倍浓缩。 </li><li>倍浓缩（同型控制）控制区域的引物进行有针对性的引物获得富集相对于除以倍浓缩（同型对照）控制未绑定的区域（ 图1）。重要的是，请注意，因为与总输入的每个样品的DNA，产生标准曲线插值的每个上述的免疫沉淀实验值，从标准曲线已经归一化到，并表示为，则该部分的总输入由机器软件。 </li><li>如果用于免疫沉淀或洗涤的缓冲区的严格性太高，它是可能的鲁棒扩增样品与特异性抗体的免疫沉淀将被观察到，而从同种型对照的扩增将不会被观察到免疫沉淀的样品。在这样的情况下，最好是由特异性抗体的同种型对照的ChIP的ChIP为目标区域和未结合的区域中减去的金额。可以计算出相对富集，将未绑定的区域（ 图3）的差异进行有针对性的区域差异。 </li></ol>

<ol>
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没有利益冲突的声明。

上述协议提供了一种可靠的方法准确地量化使用芯片的主要淋巴细胞DNA富集。在这个协议的鲁棒性的主要原因之一是列入生物复制。上述协议使用三个重复，这是独立计算的富集。然后平均的输出提供一定程度的富集和标准偏差计算提供一定程度的变异。对于每个样品，三个技术也进行消除变化的样品处理，PCR扩增等的技术重复的变化量通常远小于所观察到的不同的生物样品之间的复制。鲁棒性?...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>Sigma</td>
 <td>F-8775</td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline</td>
 <td>Hyclone</td>
 <td>SH30256.01</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Protease Inhibitor tablets</td>
 <td>Roche</td>
 <td>04693116001</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Protein G magnetic beads</td>
 <td>Active Motif</td>
 <td>101945</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>RNase A (20 mg/ml)</td>
 <td>EMD Millipore</td>
 <td>556746</td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Proteinase K (20 mg/ml)</td>
 <td>Roche</td>
 <td>03115879001</td>
 <td>Dissolve in 50 mM Tris-HCl, 10 mM CaCl2, pH 8.0</td>
 </tr>
 <tr>
 <td>Platinum Taq DNA Polymerase</td>
 <td>Invitrogen</td>
 <td>10966-034</td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>SYBR Green I</td>
 <td>Invitrogen</td>
 <td>S7567</td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>1 M Glycine</td>
 <td></td>
 <td></td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td>Cell lysis buffer (5 mM Pipes, pH 8.0; 85 mM KCl; 0.5% NP-40)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Nuclear lysis buffer (50 mM Tris, pH 8.1; 10 mM EDTA; 1% SDS)</td>
 <td></td>
 <td></td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td>ChIP dilution buffer (0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris pH 8.1; 190 mM NaCl)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Low salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris pH 8.1; 150 mM NaCl)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>High salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris pH 8.1; 600 mM NaCl)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>LiCl wash buffer (0.25 M LiCl; 1% NP-40; 1% Sodium Deoxycholate, 1 mM EDTA; 10 mM Tris pH 8.0)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA)</td>
 <td></td>
 <td></td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Elution buffer (1% SDS; 0.1 M NaHCO3)</td>
 <td></td>
 <td></td>
 <td>Prepare fresh</td>
 </tr>
 <tr>
 <td>5 M NaCl</td>
 <td></td>
 <td></td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td>0.5 M EDTA</td>
 <td></td>
 <td></td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td>1 M Tris-HCl, pH 6.5</td>
 <td></td>
 <td></td>
 <td>Store at RT</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table of Specific Reagents</td>
 </tr>
 <tr>
 <td>Clay Adams Brand Nutator</td>
 <td>Becton Dickinson</td>
 <td>Model: 421105</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnetic Stand</td>
 <td>Promega</td>
 <td>Z5342</td>
 <td></td>
 </tr>
 <tr>
 <td>Qiaquick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28106</td>
 <td></td>
 </tr>
 <tr>
 <td>Masonix Sonicator 3000</td>
 <td>QSonica</td>
 <td>Model: S3000</td>
 <td></td>
 </tr>
 <tr>
 <td>UV Spectrophotometer</td>
 <td>NanoDrop Technologies</td>
 <td>ND-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Heating Block</td>
 <td>VWR</td>
 <td>13259-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotator</td>
 <td>VWR</td>
 <td>80085-692 </td>
 <td></td>
 </tr>
 <tr>
 <td>Refrigerated bench top centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Model: Allegra X-12R</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td>Eppendorf</td>
 <td>5415 D</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table of Equipment </td>
 </tr>
 </tbody>
</table>

efficient chromatin immunoprecipitation using limiting amounts of biomass

染色质免疫沉淀技术（ChIP）是一种广泛使用的方法，用于确定不同的蛋白质与DNA的活细胞染色质的相互作用。的实例包括序列特异性DNA结合转录因子，组蛋白和其不同的修改状态，如RNA聚合酶和辅助因子，酶，DNA修复组件。尽管它的无处不在，有缺乏到最新的，详细的工作台上准备材料的准确的分析，使相互作用的定量度量方法。由于缺乏这方面的信息，并且，因为，像任何免疫沉淀的条件，必须重新优化的一组新的实验条件下，ChIP分析容易不准确或低定量的结果。 我们的协议是最终来自转录因子:DNA的相互作用1,2开创性的工作，但影响与采用了多项改进VITY和再现性难以取得的细胞类型。该协议已经被成功地使用3,4，都使用定量PCR，定量DNA浓缩，或使用以下协议的变体，一种半定量。 进行PCR扩增的材料，这种定量​​分析计算，并表示在测定中的一个限制因素。重要的控制及其他考虑因素包括使用的同种型匹配的抗体，以及评价的一个控制区域的基因组DNA，如预测由所研究的蛋白质（或预计不被束缚的间隔区不显示变化下本实验条件下）。此外，输入材料的每一个的ChIP样品的标准曲线是用来推导出在实验材料的绝对水平的富集。使用标准曲线，可以考虑到引物组之间的差异，无论它们被设计的仔细，效率也不同整个范围内的模板浓度为一个单一的引物组的分配办法。我们的协议是不同于其他5-8中，我们广泛覆盖后，分析阶段。

染色质免疫沉淀（ChIP）提出的协议控制的差异，如果有的话，通过使用一个未绑定的基因组区域的引物对，放大DNA用于PCR的金额，因此作为"负荷控制"。在图3中所示的例子中，我们已经使用鼠标Actb的基因的编码区作为未结合的蛋白，小鼠IL2作为目标区域的启动子上的兴趣和转录因子NFAT结合位点的区域。另外，几个千碱基的区域的上游或下游已知有感兴趣的蛋白质没有?...

Watch this Scientific Journal Video about Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass at JoVE.com

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

我们描述了一个可靠的方法，使用的主要的T细胞染色质免疫沉淀。该方法是建立在标准的方法，但使用一组特定的条件和试剂，提高效率有限的数量的细胞。重要的是，在数据分析阶段的详细描述。

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 ...

efficient-chromatin-immunoprecipitation-using-limiting-amounts

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.2K 次观看.  University of Utah School of Medicine. 我们描述了一个可靠的方法，使用的主要的T细胞染色质免疫沉淀。该方法是建立在标准的方法，但使用一组特定的条件和试剂，提高效率有限的数量的细胞。重要的是，在数据分析阶段的详细描述。

视频: Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

科研

生物学

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

甲基化的DNA免疫沉淀

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

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 ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

染色质免疫沉淀（ChIP）使用果蝇组织

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

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 ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

生物质的使用限制金额高效的染色质免疫沉淀

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