我们要感谢布朗和林实验室的成员，他们在参与此视频和文章批判。这项工作是由从加拿大健康研究和迈克尔史密斯健康研究基金会研究院的资金支持。

DNA的提取和样品制备从各种不同的样品（培养细胞，新鲜冰冻以及福尔马林固定石蜡包埋组织）的DNA可用于MeDIP。重要的是要使用不相关的蛋白质，如组蛋白高度纯化的DNA。同样重要的是删除尽可能多的RNA样品，因为它可以干扰DNA定量和抗体结合。 MeDIP使用的DNA的数量，范围可以从200毫微克至1微克，取决于可用的DNA量。为了证明这个协议，1微克的DNA将被使用。下面的协议，将提供高品质的双从培养细胞的双链DNA。应采用其他类型的样品中提取DNA的其他协议。 <ol><li>消化缓冲液加入400μL至1.7 mL Eppendorf管中的细胞沉淀。 </li><li>添加100微克的蛋白酶K管，并培育一夜之间在50 ° C。 </li><li>加入500μL酚pH值7和反相轻轻，但彻底混合。 </li><li>在13个000克的自旋在室温下约10分钟。 </li><li>删除一个新的管水（顶部）分数。 </li><li>重复步骤1至5 3一次。 </li><li>加入500μL1:1的酚/氯仿的pH值7和反相轻轻，但彻底混合。 </li><li>在13个000克的自旋在室温下约10分钟。 </li><li>删除一个新的管水（顶部）分数。 </li><li>添加40微克的RNA酶A孵育1小时，在37 ° C。 </li><li>加入500μL酚pH值7和反相轻轻，但彻底混合。 </li><li>在13个000克的自旋在室温下约10分钟。 </li><li>删除一个新的管水（顶部）分数。 </li><li>重复步骤13 11一次。 </li><li>加入500μL1:1的酚/氯仿的pH值7和反相轻轻，但彻底混合。 </li><li>在13个000克的自旋在室温下约10分钟。 </li><li>删除一个新的管水（顶部）分数。 </li><li>加入十分之一体积的3 M醋酸钠（40μL），拌匀。 </li><li> 100％的乙醇添加2卷（900μL），拌匀，并在-20 ° C为20分钟。 </li><li> 20分钟旋转13 000克在4 ° C。 </li><li>除去乙醇，脉冲旋转，去除残留的乙醇。 </li><li>加入500μL70％冷乙醇洗。 20分钟旋转13 000克在4 ° C。 </li><li>取出70％的乙醇，脉冲旋转，去除残留的乙醇吹打。 </li><li>空气干燥，在室温为10分钟，以去除残留的乙醇的所有痕迹开放的第沉淀。 </li><li>悬浮的DNA，在50μL灭菌dH2O一夜之间在4 ° C </li><li>使用NanoDrop分光光度计量化的DNA。一个A 260：280的比例为1.8，是理想的。 </li><li>确定100个基点阶梯琼脂糖凝胶上的DNA的质量和尺寸范围。可低至10纳克的基因组DNA 1.7％琼脂糖凝胶上运行，使用的染料，染色是高度敏感，少量的DNA，如SYBR黄​​金。 </li><li>在每一个样品的硅化管中，准备与该卷的其余部分在总体积50μL，1微克的DNA 与消毒 生署2 O </li></ol> DNA的超声 DNA超声和MeDIP协议必须执行siliconzied管，以防止非特异性结合蛋白管壁。凝胶电泳（27步）确定的DNA样本碎片程度的基础上的DNA样本的最佳超声倍。例如，从培养细胞中提取的DNA应该是非常高的分子量，并随后将需要更多的超声比这往往是部分降解的档案样本中提取DNA。如果样品是均匀的超高分子量，在这一点上，合理进行在本议定书中所描述的没有单独检查每个样品的超声。如果样品零散档案样本，就必须适应超声过程可能通过减少超声次获得300 - 100bp片段。如果您希望处理样品，部分降解，超声参数的优化，可以从利益的代表性样本进行。基于观察到的DNA片段从凝胶电泳（27步）的程度，实验者可以预先确定样品的最佳超声时间。在这里，我们描述了一种方法来获得超声与自动化超声设备使用超高分子量DNA（Bioruptor从Diagenode，UCD - 200 TM）300-1000 bp的DNA片段。 <ol><li> Biorupter水必须在4 ° C </li><li>超声用于自动设置的7分钟（30秒30秒在最大功率）。 </li><li>删除超声产品和硅化离心管1.7毫升的免疫反应（IP）的800毫微克（40μL）。 </li><li>抛开其余200纳克（10μL）作为输入（IN）的参考DNA（储存在4 ° C）。 </李&gt; </ol> IMMUNOPRECIPITATON的甲基化的DNA <ol><li>变性的，将用于在95℃水浴10分钟IP反应（800纳克）的DNA。 </li><li>酷立即在冰上。让我们的DNA完全冷却（约5分钟冰），然后再继续下一步。 </li><li>新增5微克单克隆抗体。 </li><li>添加IP缓冲液（在室温下使用整个协议）至终体积为500μL。 </li><li>旋转管持有人在2小时在4 ° C孵育。 </li><li>就在第5步完成后，准​​备磁珠洗涤（步骤6-11）。首先，重悬在小瓶珠彻底震荡。 </li><li> 30μL反应加1％的悬浮磁珠（1〜2 × 10 7）转移到一个新的硅化管（例如，8反应，如果这样做，删除9，即270μL足够珠） 。 </li><li>上放置在室温下2分钟的磁架管。 </li><li>移液器关闭上清。当去除上清，避免接触枪头对内壁珠（珠磁铁吸引）。 </li><li>取出磁铁管，并在IP缓冲区的过剩量（750μL1000μL）重悬珠。回放置在室温下2分钟的磁架管。 </li><li>重复洗一次，然后在第7步中删除原始卷重悬在IP缓冲区的水洗珠。 </li><li>加入30μL洗磁珠每个IP反应</li><li>旋转管持有人在2小时孵育4 ° C。 </li><li>孵化完成后，放在磁架管，在室温下2分钟。 </li><li>移液器关闭上清。枪头，避免接触管内壁（珠磁铁吸引）。加入500μLIP缓冲区。混合管的内容，并把2分钟的磁架。重复用500 UL IP缓冲区一次。 </li><li>删除最后一次洗涤上清液后，重悬在400消化缓冲液珠。 </li><li>治疗蛋白酶K与100微克的反应，并培育一夜之间在50 ° C。 </li></ol>免疫沉淀的DNA的分离纯化<ol><li>添加500μL1:1的酚/氯仿pH值7和涡彻底。 </li><li>在13个000克的自旋在室温下约10分钟。 </li><li>删除一个新的管水（顶部）分数。 </li><li>重复步骤1到3，如果水相和有机层之间的相间出现浑浊。 </li><li>加入1 / 10体积3 M醋酸钠（40μL）和涡。 </li><li>新增的糖原（20微克/μL）1μL和旋涡。 </li><li>添加100％的乙醇，涡旋，并在-20 ° C 20分钟2卷（1000μL）。 </li><li> 20分钟旋转13 000克在4 ° C。 </li><li>除去乙醇，脉冲旋转，去除残留的乙醇。 </li><li>加入500μL70％冷乙醇洗。涡简单地说，在13个000克的自旋为20分钟，4 ° C。 </li><li>取出70％的乙醇，脉冲旋转，去除残留的乙醇吹打。 </li><li>空气干燥，在室温为10分钟，以去除残留的乙醇的所有痕迹开放的第沉淀。 </li><li>悬浮在10μL的DNA沉淀消毒生署 2 0 。 </li></ol>通过PCR验证<ol><li>您可能会测试，以确保MeDIP程序正在执行MeDIP协议，使用正常的人类DNA（男性或女性），随后化验已知的甲基化丰富的一个区域。 </li><li>删除30％的MeDIP产品，PCR管和其他MeDIP产品的30％到另一个PCR管。 </li><li>将PCR管成10 ng的DNA中，和10纳克到另一个PCR管中的DNA。您现在应该已经设立4个独立的PCR反应。 </li><li>执行PCR扩增H19和CTRL引物如表1所示。 </li><li>准备两个mastermixes（每个引物之一），如表2所列为12.5μL总体积的PCR反应。 </li><li> Thermocycle的PCR用在表3所列的的条件。 </li><li> 5μLPCR产物在2％琼脂糖凝胶的可视化运行。 </li><li>对成功MeDIP的预期结果如表4所示。 </li></ol>表表1：H19和CTRL引物进行PCR验证。 <table border="1"><tbody><tr><td>引物</td><td>正向引物</td><td>反向引物</td><td>预期产品尺寸</td></tr><tr><td> H19 </td><td> H19_F 5&#39; - cgagtgtgcgtgagtgtgag </td><td> H19_R 5&#39; - ggcgtaatggaatgcttgaa </td><td> 174 BP </td></tr><tr><td> Ctrl（控制） </td><td> CTRL_F5&#39; - gagagcattagggcagacaaa </td><td> CTRL_R 5&#39; - gttcctcagacagccacattt </td><td> 139 BP </td></tr></tbody></table>表2。用于PCR反应Mastermixes。 <table border="1"><tbody><tr><td></td><td> H19混合（每RXN） </td><td> CTRL键组合（每RXN） </td></tr><tr><tD&gt; DDH 2 O </td><td> 6.875 </td><td> 6.875 </td></tr><tr><td> 10X缓冲</td><td> 1.25 </td><td> 1.25 </td></tr><tr><td> dNTP混合液（10毫米） </td><td> 0.25 </td><td> 0.25 </td></tr><tr><td> MgCl 2的 （50毫米） </td><td> 0.25 </td><td> 0.25 </td></tr><tr><td>引物（H19_F / R或CTRL_F / R）（10微米每个F / R） </td><td> 0.625 </td><td> 0.625 </td></tr><tr><td>白金Taq酶</td><td> 0.25 </td><td> 0.25 </td></tr></tbody></table>表3。 PCR thermocyling条件。 <table border="1"><tbody><tr><td></td><td> 95℃ </td><td> 5:00 </td></tr><tr><td rowspan="3"> 40X </td><td> 95℃ </td><td> 0:30 </td></tr><tr><td> 56℃ </td><td> 0:30 </td></tr><tr><td> 72℃ </td><td> 0:15 </td></tr></tbody></table>表4。预期的成功MeDIP PCR结果。 <table border="1"><tbody><tr><td></td><td colspan="2" align="center">模板DNA </td></tr><tr><td>底漆使用</td><td>在</td><td>知识产权</td></tr><tr><td> H19 </td><td>积极的</td><td>积极的</td></tr><tr><td>按Ctrl </td><td>积极的</td><td>负面</td></tr></tbody></table>

<ol>
	<li>Beck, S., Rakyan, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+methylome:+approaches+for+global+DNA+methylation+profiling.">The methylome: approaches for global DNA methylation profiling.</a> Trends Genet. 24, 231-237 (2008).</li><li>Lu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics,+disease,+and+therapeutic+interventions.">Epigenetics, disease, and therapeutic interventions.</a> Ageing research reviews. 5, 449-467 (2006).</li><li>Zilberman, D., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+DNA+methylation+patterns.">Genome-wide analysis of DNA methylation patterns.</a> Development. 134, 3959-3965 (2007).</li><li>Feinberg, A. P., Tycko, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+cancer+epigenetics.">The history of cancer epigenetics.</a> Nature reviews. 4, 143-153 (2004).</li><li>Weber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution,+silencing+potential+and+evolutionary+impact+of+promoter+DNA+methylation+in+the+human+genome.">Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome.</a> Nat Genet. 39, 457-466 (2007).</li><li>Weber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome-wide+and+promoter-specific+analyses+identify+sites+of+differential+DNA+methylation+in+normal+and+transformed+human+cells.">Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells.</a> Nat Genet. 37, 853-862 (2005).</li><li>Wilson, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenomics:+mapping+the+methylome.">Epigenomics: mapping the methylome.</a> Cell Cycle. 5, 155-158 (2006).</li><li>Gazin, C., Wajapeyee, N., Gobeil, S., Virbasius, C. M., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+elaborate+pathway+required+for+Ras-mediated+epigenetic+silencing.">An elaborate pathway required for Ras-mediated epigenetic silencing.</a> Nature. 449, 1073-1077 (2007).</li><li>Karpinski, P., Sasiadek, M. M., Blin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+epigenetic+patterns+in+the+etiology+of+gastrointestinal+cancers.">Aberrant epigenetic patterns in the etiology of gastrointestinal cancers.</a> Journal of applied. 49, 1-10 (2008).</li><li>Maekawa, M., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics:+relations+to+disease+and+laboratory+findings.">Epigenetics: relations to disease and laboratory findings.</a> Current medicinal chemistry. 14, 2642-2653 (2007).</li><li>Vucic, E. A., Brown, C. J., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+of+cancer+progression.">Epigenetics of cancer progression.</a> Pharmacogenomics. 9, 215-234 (2008).</li><li>Egger, G., Liang, G., Aparicio, A., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+in+human+disease+and+prospects+for+epigenetic+therapy.">Epigenetics in human disease and prospects for epigenetic therapy.</a> Nature. 429, 457-463 (2004).</li><li>Jones, P. A., Baylin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+epigenetic+events+in+cancer.">The fundamental role of epigenetic events in cancer.</a> Nat Rev Genet. 3, 415-428 (2002).</li><li>Fraga, M. F., Esteller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+a+profile+of+methods+and+applications.">DNA methylation: a profile of methods and applications.</a> Biotechniques. 33, 632-649 (2002).</li></ol>

有一个日益认识到疾病的DNA甲基化起着重要作用，因此发展的分析来衡量这一修改正在成为越来越重要的3，12，13 。 MeDIP技术是筛选适合的工具，同时在全基因组位点特异性的第6级 ， 7。这种技术提供了一个快速查看和使用数量有限的起始DNA的DNA甲基化水平，为便于比较不同来源之间允许。使用该MeDIP产品的下游应用领域包括各种芯片的全基因组CpG岛寡核苷酸阵列，直接P...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Biorupter sonicator</td>
<td>Tool</td>
<td>Diagenode</td>
<td>UCD-200 TM</td>
<td>&nbsp;</td>
<tr>
<td>1.7ml SafeSeal Microcentrifuge Tubes</td>
<td>Other</td>
<td>Sorenson BioScience</td>
<td>11510</td>
<td>&nbsp;</td>
<tr>
<td>ND 3300 Spectrophotometer</td>
<td>Tool</td>
<td>NanoDrop</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Primary Antibody: Anti-5'-methylcytosine mouse mAb</td>
<td>Reagent</td>
<td>CalBiochem</td>
<td>162 33 D3</td>
<td>&nbsp;</td>
<tr>
<td>Secondary Antibody: Dynabeads M-280 Sheep anti-mouse IgG</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>112-01D</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Tube Rack</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>CS15000</td>
<td>&nbsp;</td>
<tr>
<td>Mini LabRoller</td>
<td>Tool</td>
<td>Labnet International</td>
<td>H5500</td>
<td>&nbsp;</td>
<tr>
<td>IP Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100. Stored at room temperature</td>
</tr>
<tr>
<td>Digestion Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM Tris pH8.0, 100 mM EDTA, 0.5% SDS, 50 mM NaCl</td>
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methylated dna immunoprecipitation

识别DNA甲基化模式是表观遗传学的研究的一个常见的​​程序，作为甲基化是已知的基因表达有显着影响，并与参与的正常发展，以及疾病<sup&gt; 1-4</sup&gt;。因此，能力，区分甲基化的DNA和非甲基化的DNA甲基化谱等研究产生至关重要的。甲基化DNA免疫共沉淀（MeDIP）是一个有效的技术甲基化的DNA的提取样本的利益<sup&gt; 5-7</sup&gt;。低至200纳克的DNA样本是足够的抗体或免疫沉淀（IP），反应。 DNA是超声在300-1000 BP大小不等的片段，并划分为免疫沉淀（IP）和输入（IN）部分。 IP DNA随后热变性，然后用反5&#39;mC培养，使单克隆抗体结合甲基化的DNA。在此之后，具有亲和力的抗体二抗磁珠添加，并且孵育。这些珠联抗体结合的单克隆抗体用于第一步。势必抗体复合物（甲基化的DNA）的DNA是分开使用磁铁拉配合物的解决方案，其余的DNA。使用IP缓冲区的几个清洗，然后进行删除未绑定，非甲基化的DNA。甲基化的DNA /抗体复合物，然后用蛋白酶K消化，消化离开只有甲基化的DNA完整的抗体。富集的DNA纯化酚：氯仿抽提去除蛋白质的物质，然后沉淀和再悬浮在水中以备后用。 PCR技术可用于分析知识产权的扩增产物，在已知缺乏的地区，已知含有甲基化序列的DNA，来验证的MeDIP程序的效率。然后可以用于纯化的甲基化的DNA位点特异性（PCR）或全基因组（芯片和测序）甲基化研究，并在结合其他的研究工具，如基因表达谱和阵列比较基因组杂交应用时尤其有用（ CGH）<sup&gt; 8</sup&gt;。到DNA甲基化的进一步调查发现的新的后生目标，这反过来，可能aberrantly甲基化的DNA特征的疾病，如癌症，在发展中国家的新的治疗和预后的研究工具非常有用<sup&gt; 2，4，9-11</sup&gt;。

Watch this Scientific Journal Video about Methylated DNA Immunoprecipitation at JoVE.com

Methylated DNA Immunoprecipitation

这个视频演示的甲基化的DNA免疫（MeDIP）协议。 MeDIP是有选择性地从基因组DNA样本中提取5 - 甲基胞嘧啶甲基化的DNA片段具有特异性抗体（抗- 5 MC）为期两天的过程。

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 ...

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JoVE Journal

Biology

23.2K 次观看.  BC Cancer Research Centre. 这个视频演示的甲基化的DNA免疫（MeDIP）协议。 MeDIP是有选择性地从基因组DNA样本中提取5 - 甲基胞嘧啶甲基化的DNA片段具有特异性抗体（抗- 5 MC）为期两天的过程。

视频: Methylated DNA Immunoprecipitation

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

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

抗体分析的荧光素酶免疫沉淀系统（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA的稳定同位素探测（DNA的SIP）

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM:免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

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 ...

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 ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

迭代优化的DNA双链的SeqA-DNA复合物的结晶

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

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 ...

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 ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...