Name: chromatin immunoprecipitation assay using micrococcal nucleases in mammalian cells
Uploaded: 2019-05-10T14:30:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells at JoVE.com

这项研究得到了基因科技对希望之城的版税的支持。这项工作没有得到国家卫生研究院的全部或部分支持。

1. 试剂制备
<ol><li>制造18.5%的甲醛(PFA)溶液。在 50 mL 锥形塑料管中加入 0.925 g 的 PFA、4.8 mL 的水(在整个协议中使用超净化水)和 35 μL 的 1 M KOH。用微波炉将盖子紧紧合住,在 400-600 mL 玻璃烧杯中加热管,其中包含约 200 mL 的水。在水开始沸腾之前取出管子,并涡旋管以溶解 PFA。让 PFA 冷却到室温并储存在冰上。 注:重复加热和混合,直到 PFA 完全溶解。在交联之前立即准备 PFA 解决方案(步?...

<ol>
	<li>Nikolov, D. B., Burley, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+II+transcription+initiation:+a+structural+view.">RNA polymerase II transcription initiation: a structural view.</a> Proceedings of the National Academy of Sciences of the United States of America. 94 (1), 15-22 (1997).</li><li>Strahl, B. D., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+language+of+covalent+histone+modifications.">The language of covalent histone modifications.</a> Nature. 403 (6765), 41-45 (2000).</li><li>Hellman, L. M., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+(EMSA)+for+detecting+protein-nucleic+acid+interactions.">Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.</a> Nature protocols. 2 (8), 1849-1861 (2007).</li><li>Jackson, V., Chalkley, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+whole-cell+fixation+to+visualize+replicating+and+maturing+simian+virus+40:+identification+of+new+viral+gene+product.">Use of whole-cell fixation to visualize replicating and maturing simian virus 40: identification of new viral gene product.</a> Proceedings of the National Academy of Sciences of the United States of America. 78 (10), 6081-6085 (1981).</li><li>Jackson, V., Chalkley, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+isolation+of+replicative+chromatin:+selective+deposition+of+histone+on+both+new+and+old+DNA.">A new method for the isolation of replicative chromatin: selective deposition of histone on both new and old DNA.</a> Cell. 23 (1), 121-134 (1981).</li><li>Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+state+of+chromatin+immunoprecipitation.">The current state of chromatin immunoprecipitation.</a> Molecular Biotechnology. 45 (1), 87-100 (2010).</li><li>Ausubel, R. B., Kingston, R. E., Moore, D. D., Smith, J. A., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Genomic+DNA.">Preparation of Genomic DNA.</a> Short Protocols in Molecular Biology. , (1992).</li><li>Crouse, J., Amorese, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+Precipitation:+Ammonium+Acetate+as+an+Alternative+to+Sodium+Acetate.">Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate.</a> Focus. 9 (2), 3-5 (1987).</li><li>Zeugin, J. A., Hartley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+Precipitation+of+DNA.">Ethanol Precipitation of DNA.</a> Focus. 7 (4), 1-2 (1985).</li><li>Barski, A., 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=High-resolution+profiling+of+histone+methylations+in+the+human+genome.">High-resolution profiling of histone methylations in the human genome.</a> Cell. 129 (4), 823-837 (2007).</li><li>Guenther, M. G., Levine, S. S., Boyer, L. A., Jaenisch, R., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chromatin+landmark+and+transcription+initiation+at+most+promoters+in+human+cells.">A chromatin landmark and transcription initiation at most promoters in human cells.</a> Cell. 130 (1), 77-88 (2007).</li><li>Louie, M. C., 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=Androgen-induced+recruitment+of+RNA+polymerase+II+to+a+nuclear+receptor-p160+coactivator+complex.">Androgen-induced recruitment of RNA polymerase II to a nuclear receptor-p160 coactivator complex.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (5), 2226-2230 (2003).</li><li>Liu, X., 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=Positive+feedback+loop+mediated+by+protein+phosphatase+1alpha+mobilization+of+P-TEFb+and+basal+CDK1+drives+androgen+receptor+in+prostate+cancer.">Positive feedback loop mediated by protein phosphatase 1alpha mobilization of P-TEFb and basal CDK1 drives androgen receptor in prostate cancer.</a> Nucleic Acids Research. 45 (7), 3738-3751 (2017).</li><li>Zhao, H., 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=Identification+of+valid+reference+genes+for+mRNA+and+microRNA+normalisation+in+prostate+cancer+cell+lines.">Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines.</a> Scientific reports. 8 (1), 1949 (2018).</li><li>Nelson, J. D., Denisenko, O., Bomsztyk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+the+fast+chromatin+immunoprecipitation+(ChIP)+method.">Protocol for the fast chromatin immunoprecipitation (ChIP) method.</a> Nature. 1 (1), 179-185 (2006).</li><li>Gade, P., Kalvakolanu, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+assay+as+a+tool+for+analyzing+transcription+factor+activity.">Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity.</a> Methods in molecular biology. 809, 85-104 (2012).</li><li>Lund, E. G., Duband-Goulet, I., Oldenburg, A., Buendia, B., Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+features+of+lamin+A-interacting+chromatin+domains+mapped+by+ChIP-sequencing+from+sonicated+or+micrococcal+nuclease-digested+chromatin.">Distinct features of lamin A-interacting chromatin domains mapped by ChIP-sequencing from sonicated or micrococcal nuclease-digested chromatin.</a> Nucleus. 6 (1), 30-39 (2015).</li><li>Dahl, J. A., Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+micro+chromatin+immunoprecipitation+assay+(microChIP).">A rapid micro chromatin immunoprecipitation assay (microChIP).</a> Nature protocols. 3 (6), 1032-1045 (2008).</li><li>Yamakawa, T., Waer, C., Itakura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AT-rich+interactive+domain+5B+regulates+androgen+receptor+transcription+in+human+prostate+cancer+cells.">AT-rich interactive domain 5B regulates androgen receptor transcription in human prostate cancer cells.</a> Prostate. 78 (16), 1238-1247 (2018).</li></ol>

虽然声波通常用于获得零碎的染色质,但识别可重现的条件既耗时又繁琐。在此协议中,我们使用MNase消化,因为酶消化应该更容易识别可重复的条件。在MNase消化后,需要一个短暂的声波步骤(参见步骤2.2)来破坏细胞膜并释放消化的染色质。因此,我们协议中的声波功率应该尽可能低。我们对所有使用的细胞使用相同的声波条件(参见图1和表1)来获得膜的完全分解。
...

哺乳动物细胞的基因表达是严格而动态的调节,转录是关键步骤之一。基因转录主要受转录因子和组蛋白的调节。转录因子是一种蛋白质,它与特定的DNA序列结合并控制基因转录。这些因素要么促进或抑制RNA聚合酶II(PolII)的招募,它启动从基因组DNA作为模板1的mRNA合成。组蛋白修饰,如乙酰化和甲基化组蛋白尾残留物正和消极影响基因转录通过改变染色质结构2。由于基因表达的变化会影响细胞上下文,因此有必要研究转录的分子机制。
迄今为止,已有几种研究基因转录调控的方法可用。电泳移动移位测定(EMSA),也称为凝胶移位测定,用于分析蛋白质-DNA相互作用3。从感兴趣的细胞中孵育出一种放射性同位素(例如,32 P)标记的DNA探针,并在聚丙烯酰胺凝胶上电泳。其自动放射图显示,DNA-蛋白质复合物的迁移速度比凝胶中的探针慢。在存在针对蛋白质的抗体的情况下,DNA-蛋白质-抗体复合物在凝胶中迁移的速度比DNA-蛋白质复合物要慢。这个超移带揭示了DNA和蛋白质之间的特定结合。然而,EMSA只确定细胞无细胞系统中的特定DNA-蛋白质相互作用,因此,该相互作用是否控制活细胞的转录仍不得而知。瞬态报告器测定,通常称为荧光酶报告器测定,是为解决细胞中的基因表达调节而开发的。通常 , 感兴趣的基因的上游基因组区域入到报告质粒中 , 瞬时转染成细胞 , 并测量报告者的活动。各种缺失突变体允许识别负责基因调控的区域。尽管报告员检测是识别转录因子和控制转录的结合DNA序列的有用工具,但这种方法有一个主要缺点,即报告质粒不含染色质结构,不能反映"真实"转录机械。此外,组蛋白修改的更改不能由系统确定。
染色质免疫沉淀(ChIP)方法的发展是基于杰克逊和查克利的报告,"全细胞"固定与甲醛保留染色质结构4,5。自那时以来,许多相关的技术已经开发和改进6。在ChIP测定中,细胞用甲醛固定,以交叉连接DNA和蛋白质。染色质是支离破碎的,然后用感兴趣的抗体进行免疫沉淀。免疫复合物被洗净,DNA被纯化。PCR扩增与引基因靶向基因组的特定区域揭示感兴趣的蛋白质在基因组的占用。
虽然ChIP是识别蛋白质(如转录因子)和修饰组蛋白与DNA相互作用的有力工具,但该方法在实践中涉及一些困难,如染色质破碎步骤。声波已广泛应用于剪切染色质;但是,识别可重现的条件很麻烦。微球核酸酶(MNase)治疗是染色质剪切的替代方法。MNase是一种内源性酶,可消化双链、单链、圆形和线性DNA和RNA。确定最佳染色质分片的条件(包括染色质和酶的量、温度和孵育时间)相对容易。我们修改和简化了现有的协议,并建立了一个简单易重用的方法。本文提供了在哺乳动物细胞中使用MNase的ChIP测定方案。

<table border="0" cellpadding="0" cellspacing="0" style="width:1643px;" width="1642">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">0.5 M EDTA (pH 8.0)</td>
			<td style="width:173px;">Thermo Scientific</td>
			<td style="width:119px;">AM9010</td>
			<td style="width:793px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">2 M KCl</td>
			<td style="width:173px;">Thermo Scientific</td>
			<td style="width:119px;">AM9010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">2X iQ SYBR Green supermix</td>
			<td style="width:173px;">Bio-Rad</td>
			<td align="right" style="width:119px;">1706862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">5 M NaCl</td>
			<td style="width:173px;">Thermo Scientific</td>
			<td style="width:119px;">AM9010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">50 bp DNA ladder</td>
			<td style="width:173px;">New England Biolabs</td>
			<td style="width:119px;">N3236S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:557px;">Agarose</td>
			<td style="width:173px;">Research Product International</td>
			<td style="width:119px;">A20090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Branched octylphenoxy poly(ethyleneoxy)ethanol</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">I8896</td>
			<td>IGEPAL CA-630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">ChIP-grade protein G magnetic beads</td>
			<td style="width:173px;">Cell signaling technology</td>
			<td style="width:119px;">9006S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Chromatin Immunoprecipitation (ChIP) Dilution Buffer</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">20-153</td>
			<td>Buffer composition: 0.01% SDS, 1.1% Triton X- 100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Gel Loading Dye Purple (6X)</td>
			<td style="width:173px;">New England Biolabs</td>
			<td style="width:119px;">B7024S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Glycine</td>
			<td style="width:173px;">Bio-Rad</td>
			<td style="width:119px;">161-0724</td>
			<td>Electropheresis grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Glycogen</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">G1767</td>
			<td>19-22 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x)</td>
			<td style="width:173px;">Thermo Scientific</td>
			<td style="width:119px;">78445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">High Salt Immune Complex Wash Buffer</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">20-155</td>
			<td>Buffer composition: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Histone H3K4me3 antibody (pAb)</td>
			<td style="width:173px;">Active Motif</td>
			<td style="width:119px;">39915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">LiCl Immune Complex Wash Buffer</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">20-156</td>
			<td>Buffer composition: 0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Low Salt Immune Complex Wash Buffer</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">20-154</td>
			<td>Buffer composition: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Magna GrIP Rack (8 well)</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">20-400</td>
			<td style="width:793px;">Any kind of magnetic separation stands that are compatible with a 1.5 mL tube is fine.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Micrococcal nuclease</td>
			<td style="width:173px;">New England Biolabs</td>
			<td style="width:119px;">M0247S</td>
			<td>comes with 10 x buffer (500 mM Tris-HCl, 50 mM CaCl2, pH 7.9 @ 25 &#176;C) and 100 x BSA (10 mg/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">NaHCO3</td>
			<td style="width:173px;">JT Baker</td>
			<td style="width:119px;">3506-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Normal rabbit IgG</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">12-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">PIPES</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">P6757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Proteinase K</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">3115887001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Real-time PCR system</td>
			<td style="width:173px;">Bio-Rad</td>
			<td style="width:119px;">CFX96, C1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">RNA pol II CTD phospho Ser5 antibody</td>
			<td style="width:173px;">Active Motif</td>
			<td style="width:119px;">39749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">SDS</td>
			<td style="width:173px;">Boehringer Mannheim</td>
			<td style="width:119px;">100155</td>
			<td>Electropheresis grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">sodium acetate</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">S5636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Sonicator equipped with a microtip probe</td>
			<td style="width:173px;">QSONICA</td>
			<td style="width:119px;">Q700</td>
			<td style="width:793px;">Any kind of sonicators that are compatible with a 1.5 mL tube is fine.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)</td>
			<td style="width:173px;">Thermo Scientific</td>
			<td style="width:119px;">15593031</td>
			<td>pH 8.05</td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation assay using micrococcal nucleases in mammalian cells

为了表达生物体内的细胞表型,活细胞相应地执行基因表达,转录程序在基因表达中起着核心作用。细胞转录机及其染色质修饰蛋白协调以调节转录。为了在分子水平上分析转录调控,可以使用几种实验方法,如电泳移动移位、瞬态报告器和染色质免疫沉淀(ChIP)测定。本文详细介绍了经过修饰的ChIP测定,因为它具有直接显示组蛋白修饰和细胞中蛋白质和DNA之间的相互作用的优点。成功的 ChIP 测定的关键步骤之一是染色质剪切。虽然声波常用于剪切染色质,但很难识别可重现的条件。我们采用微球核酸酶(MNase)的酶消化,而不是通过声波剪切染色质,从而获得更可重复的结果。在本文中,我们使用MNase提供一个简单的ChIP测定协议。

消化染色质是ChIP测定的重要步骤之一。我们使用MNase消化染色质,以获得核小体寡聚物的混合物。在MNase消化步骤中,MNase可以通过核膜和消化染色质。然而,消化的染色质不能穿过膜,并留在核中。要从核中释放消化的染色质,需要短暂的声波。图1A显示了VCaP细胞悬浮液声波前后的微照片。在没有声波的情况下,细胞结构保持不变,表明染色质存在于核中。短暂的声波会破坏细胞结?...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells at JoVE.com

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

染色质免疫沉淀(ChIP)是了解基因调控的分子机制的有力工具。然而,该方法在通过机械剪切获得可重复染色质碎片方面遇到了困难。在这里,我们为使用酶消化的ChIP测定提供了改进的协议。

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

在该协议中，我们可以识别蛋白质和DNA之间的相互作用，揭示基因的转录调控。这有助于理解细胞中的分子机制。我们修改预先存在的协议，然后建立一个简单明了的检测协议。您只需按照第一步的协议进行，即可成功完成测定。为了准备交联染色质，在烟罩中，将四毫升1%VCaP细胞培养基和0.229毫升18.5%PFA的0.229毫升添加到6厘米的培养皿中。轻轻旋转盘，均匀分布 PFA。在室温下孵育整整10分钟。然后，在菜中加入0.47毫升1.25摩尔甘油溶液，并在室温下孵育5分钟，停止进一步的交联。根据手稿清洗细胞后，刮取细胞，将细胞悬浮液转移到1.5毫升微离心管。在室温下将带细胞悬浮液的管子在3000次g下离心5分钟，以回收细胞颗粒。使用移液器完全拆下 PBS。记录每个管的细胞数，并在零下80摄氏度储存细胞颗粒。要进行细胞解解，首先在冰上解冻储存的VCAP细胞颗粒。将 300 微升的已准备好的 ChIP 细胞解液缓冲液添加到每个管中，并辅以 PIC，并彻底重新填充颗粒。旋转管15秒，在冰上孵化悬浮液10分钟。在摄氏4度下以9000倍的温度下离心，3分钟。完全去除上流剂，并加入300微升的MNase消化缓冲液，以重新暂停颗粒。在1.5毫升管中用MNase消化缓冲液稀释MNase，每微升产生50个凝胶单位。将稀释的MNase加入悬浮液中，在37摄氏度下孵育10分钟。每 2 1~2 分钟通过反转设置混合。为了终止MNase消化，在pH8处加入30微升0.5摩尔EDTA，并短暂加入涡流。在冰上孵育5分钟后，在摄氏4度下以9000次g离心，5分钟。完全去除上流液，将颗粒重新在 300 微升的已准备的 ChIP 稀释缓冲液中，并辅以 PIC。然后，在配备微提示探头的声波器上，将声波条件设置为振幅2，处理时间15秒，脉冲开5秒，脉冲关闭30秒。在冰上对悬浮液进行声波化。绘制悬架的一微升，并点到滑动玻璃上。在显微镜下，观察以确保细胞结构几乎被打破。在4摄氏度下以9000倍g离心管10分钟，然后将上流水线转移到新的1.5毫升微离心管中。将消化的染色质的20微升保存在1.5毫升的螺丝管中，用于进一步治疗，并在零下80摄氏度下储存其余。首先，在每个1.5毫升的螺杆管中，加入75微升水、4微升五摩尔氯化钠和1微升蛋白酶K.To去除蛋白质和DNA之间的交联，将20微升的消化铬素从每个MNase消化条件添加到螺杆管中。紧闭盖子，完全混合，并在65摄氏度过夜孵育管。早晨，在将管子离心2到3000次g后，加入100微升PCI。大力旋转，形成乳液。在室温下，以最高速度再次离心管 30 秒。然后，准备两个1.5毫升的微离心管每个条件。在一个管中加入100微升PCI。在pH 5.2下加入10微升三摩尔醋酸钠，将两微升糖原加入另一根管中。从从离心机中取出的管子中，小心地绘制含有DNA的上相，并将其添加到含有PCI的管中。大力旋转，并在室温下以最高速度离心管30秒。接下来，将上相转移到含有醋酸钠和糖原的管中。加入250微升乙醇，通过倒置混合。在室温下孵育10分钟。在4摄氏度下以最高速度将管子离心30分钟。确认管底的颗粒。小心地去除上一液，以免干扰颗粒，并加入500微升70%乙醇。在4摄氏度下以最高速度将管子离心5分钟。完全取出上一液，在室温下干燥颗粒约五分钟。然后，加入20微升TE溶解颗粒。使用紫外分光光度计测量DNA浓度。制备消化的染色质后，将所有样品解冻在冰上。加入1200微升的已准备的ChIP稀释缓冲液，辅以PIC稀释消化的染色质，浓度为每500微升5微克。将稀释的消化染色质的五微升加入 1.5 毫升的螺杆管作为输入样品。样品储存在零下80摄氏度。将稀释后的染色质每层加入500微升，加入1.5毫升的螺杆，作为免疫沉淀样品。在每个管中加入两微克抗体，然后关闭瓶盖。在四摄氏度下孵育管子，在摇台上轻轻混合。早晨，在每个管中加入30微升ChIP级蛋白G磁珠。在四摄氏度下再次孵育管子，用温和的混合孵育两个小时。之后，以2至3000倍g的短暂速度将管子旋转，将管放在含有镉磁铁的聚乙烯机架中一分钟。然后，小心地通过吸气去除上一杯。接下来，在每个管中加入0.5毫升的低盐免疫复合洗涤缓冲液。短暂地旋转管子以分散珠子。在四摄氏度下孵育管子五分钟，在摇台上轻轻混合。根据手稿，使用高盐免疫复合物和氯化锂免疫复合物重复洗涤。完全去除剩余的上流液后，向管中加入150微升洗脱缓冲液。旋转管完全分散珠子。关闭盖子，在65摄氏度下孵育管子30分钟。要彻底分散珠子，每五分钟进行一次反转混合。在孵育过程中，准备一个1.5毫升的螺丝管，并加入6微升的五摩尔氯化钠和2微升的蛋白酶K.Thaw以前在冰上制备的1%输入样品。在输入样品中加入150微升洗脱缓冲液、6微升五摩尔氯化钠和2微升蛋白酶K。孵育含有珠子的管子后，旋转下来。将管放在磁架中一分钟，将上清液转移到含有氯化钠和蛋白酶的螺杆管上。没有声波，细胞结构保持不变，表明染色质存在于核中。短暂的声波会打破细胞结构。交联染色质由VCaP细胞中产生，并用不同量的MNase进行消化。在不添加MNase的情况下，出现了分子量非常高的涂片模式。MNase的加入给出了一个阶梯图案，这表明MNase消化了内分体。只有产生高达900 bp的染色质片段的条件才能被认为是适当的消化，而不适当的消化模式显示过度消化，主要导致单核糖体生产。VCaP细胞中染色质的消化模式表明染色质的消化过程。H3K4甲基基率最高的是AR-TSS上游的0.5千基和1个千基。但是，位于AR-TSS下游19千基和8 kb和12千基的地区，H3K4甲基基3的占用率很小，表明这些区域可以用作负区域。确定MNase消化条件是最重要的。在每个实验中保持细胞数、缓冲量和酶量。必须为每种单元格类型标识条件。我们可以对因子和染色质均质捕获的全球占用进行 ChIP 测序，以确定致发基因组病变之间的相互作用。该方法可以揭示转录的过程机制。ChIP 检测是调查转录调控的有力工具，但繁琐且不可重复。我们的方法鼓励研究人员进行常规和容易的测定。

Research

JoVE Journal

Genetics

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

科研

遗传学

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

染色质免疫沉淀（ChIP）在小鼠T细胞系

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

从酿酒酵母中组蛋白修饰的染色质沉淀 (芯片)

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

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 ...

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

使用染色质免疫沉淀和高通量测序（ChIP-seq）发现沙门氏菌生物膜中的CsgD监管目标

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

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

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

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 ...