在这个手稿的工作是由放射肿瘤学系和Huntsman癌症研究所和卫生拨款的国家研究所（R01-CA188520），以SB的资金支持。我感谢丹尼尔·约翰逊和史蒂芬·贝内特在我的实验室演示的协议和解释它的好处。我很感谢马赫什Chandrasekharan博士（Huntsman癌症研究所）对稿件批评意见。

1.染色质免疫沉淀（ChIP）以下BrdU标记<ol><li>培养NIH3T3细胞在DMSO或HDAC1,2选择性抑制剂的存在下如前面6所述。 </li><li>以下用DMSO或HDAC1,2选择性抑制剂治疗，在无菌组织培养罩添加BrdU的细胞中。孵育2×10 6个NIH3T3细胞接种在10ml的NIH3T3介质溴脱氧尿苷的20μM的最终浓度（BrdU）标记60分钟在无菌37℃组织培养箱中。 </li><li>交联细胞内蛋白质的DNA直接添加甲醛到培养基在1％的终浓度。孵育...

<ol>
	<li>Podhorecka, M., Skladanowski, A., Bozko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX+Phosphorylation:+Its+Role+in+DNA+Damage+Response+and+Cancer+Therapy.">H2AX Phosphorylation: Its Role in DNA Damage Response and Cancer Therapy.</a> J nucleic acids. , (2010).</li><li>Groth, A., Rocha, W., Verreault, A., Almouzni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+challenges+during+DNA+replication+and+repair.">Chromatin challenges during DNA replication and repair.</a> 128, 721-733 (2007).</li><li>Demeret, C., Vassetzky, Y., Mechali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+remodelling+and+DNA+replication:+from+nucleosomes+to+loop+domains.">Chromatin remodelling and DNA replication: from nucleosomes to loop domains.</a> Oncogene. 20, 3086-3093 (2001).</li><li>Bhaskara, S., 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=Deletion+of+histone+deacetylase+3+reveals+critical+roles+in+S+phase+progression+and+DNA+damage+control.">Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control.</a> Mol Cell. 30, 61-72 (2008).</li><li>Bhaskara, S., Hiebert, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+histone+deacetylase+3+in+maintenance+of+genome+stability.">Role for histone deacetylase 3 in maintenance of genome stability.</a> Cell Cycle. 10, 727-728 (2011).</li><li>Bhaskara, S., 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=Histone+deacetylases+1+and+2+maintain+S-phase+chromatin+and+DNA+replication+fork+progression.">Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression.</a> Epigenetics Chromatin. 6, 27 (2013).</li><li>Bhaskara, S., 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=Hdac3+is+essential+for+the+maintenance+of+chromatin+structure+and+genome+stability.">Hdac3 is essential for the maintenance of chromatin structure and genome stability.</a> Cancer Cell. 18, 436-447 (2010).</li><li>Johnson, D. 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B., Turner, B. M., Almouzni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplication+and+maintenance+of+heterochromatin+domains.">Duplication and maintenance of heterochromatin domains.</a> J Cell Biol. 147, 1153-1166 (1999).</li><li>Alabert, C., Groth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+replication+and+epigenome+maintenance.">Chromatin replication and epigenome maintenance.</a> Nat Rev Mol Cell Biol. 13, 153-167 (2012).</li><li>Clapier, C. R., Cairns, B. 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在这个手稿中描述的协议是一个相对快速的方法以证明对新复制或新生的DNA的蛋白质或它们的翻译后修饰的形式的存在。此外，这种技术允许一个以测量的蛋白质或它与新生的DNA修饰形式的关联-解离动力学。这种技术是相辅相成的优雅iPOND技术13。在iPOND技术，新合成的DNA标记用乙酸乙酯脱氧尿苷（EDU）。生物素结合物，然后用点击化学添加到EDU。生物素标记的新​​生DNA，然后用链霉珠?...

有缺陷的DNA修复和/或DNA复制是基因组不稳定性，这可以触发细胞死亡的一个主要原因。单个未修复的双链断裂足以导致细胞死亡1。染色质组织都复制期间瞬时改变和修2,3，和故障维护表观遗传信息在这些过程将导致威胁基因组的完整性。 HDAC3或HDAC1,2功能丧失阻碍S期的进展，DNA复制和修复，导致基因毒性应激（DNA损伤）和细胞死亡4-9。因此，它是一个实际的策略，利用选择性HDAC抑制剂破坏复制，修复和染色质在癌细胞和引起DNA损伤，这反过来又可以停止生长，诱导细胞死亡选择性地迅速增长的癌细胞。 期间DNA复制，染色质快速拆卸和重新组装下列DNA重复。新学年nthesized组蛋白H4设置在K5和K12残基（H4K5K12ac）和以下沉积乙酰到染色质，它们被迅速由组蛋白脱乙酰酶（HDACs）10,11脱乙酰。细胞未能保持由组蛋白去乙酰化新生染色质完整性可导致叉崩溃，这反过来可导致DNA损伤和细胞死亡。我们最近发现，HDAC1,2选择性抑制增加组蛋白乙酰化（H4K5ac，H4K12ac和H4K16ac）并抑制SMARCA5染色质remodeler活动上新生染色质，这与减少的复制叉进展，增加的叉崩溃和增加的复制压力诱导的DNA损伤6 。因此，HDAC抑制剂治疗可以改变新生染色质结构，以非常快速地触发DNA损伤和死亡癌细胞中，因为这些细胞周期迅速并通过S相通许多次。据了解的HDAC如何发挥作用来调节组蛋白乙酰化和蛋白质宾迪因此，重要纳克维持在哺乳动物细胞中DNA的复制。 定量地测量组蛋白乙酰化和DNA复制过程中与新生的DNA分子上SMARCA5染色质remodeler的量，我们设计了一种改性ChIP实验称为BrdU的芯片插槽西方技术。使用槽孔以下的所希望的蛋白质或组蛋白修饰染色质免疫沉淀（ChIP），新生的DNA的量的沉淀样品中可以使用的BrdU标记的ChIP的DNA印迹分析来检测（使用胸苷类似物，BrdU阳性）转移到膜印迹装置。使用这种技术，我们发现，H4K16ac（参与染色质包装的标记）和ISWI家族成员SMARCA5（染色质SWI / SNF相关的基质相关的肌动蛋白依赖调节剂）的染色质remodeler与在S期细胞新生DNA的关联6。我们还发现，新生H4K16ac标记通过HDAC1,2 DNA复制6中脱乙酰。 H4K16ac抑制在ISWI家族成员SMARCA5 12的染色质remodeler活动。因而，使用的BrdU片插槽西方技术中，我们可以连接为HDAC1,2的功能在DNA复制过程中调节染色质重塑。因此，尿嘧啶芯片插槽，Western印迹技术是一种有效的方法来定量测量绑定到新生的DNA或蛋白质的翻译后修饰的结合和解离动力学。

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-BrdU (westerns)</td>
			<td style="width:107px;">BD Biosciences</td>
			<td style="width:119px;">B555627</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;">Anti-SMARCA5</td>
			<td>Abcam</td>
			<td style="width:119px;">ab3749</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Anti-H4K16ac</td>
			<td style="width:107px;">Active Motif</td>
			<td>39167</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Zeta-Probe GT Membrane</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">162-0197</td>
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			<td height="21" style="height:21px;width:216px;">Formaldehyde</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:119px;">BP531-500</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Protein A agarose</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;">16-156</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Rnase A</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:119px;">19101</td>
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			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">P4850</td>
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			<td height="21" style="height:21px;width:216px;">PCR Purification Kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:119px;">28106</td>
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			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">G7403</td>
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			<td height="21" style="height:21px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:107px;">Roche</td>
			<td style="width:119px;">11836170001</td>
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			<td height="21" style="height:21px;width:216px;">BrdU</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">B9285</td>
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			<td height="21" style="height:21px;width:216px;">Rabbit IgG&#160;</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;">12-370</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">S-3014</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Triton-X-100</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">93443</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">NP40</td>
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			<td style="width:119px;">19628</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Sodium deoxycholate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">D6750</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">S-4019</td>
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			<td height="42" style="height:42px;width:216px;">Ethanol</td>
			<td style="width:107px;">Decon Laboratories</td>
			<td style="width:119px;">04-355-222</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">DEPC-treated water</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">95284</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:119px;">BP-152-5</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:107px;">Ambion</td>
			<td style="width:119px;">AM9820</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Sodium hydroxide</td>
			<td style="width:107px;">Mallinckrodt GenAR&#160;</td>
			<td style="width:119px;">MAL7772-06</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">20X SSC</td>
			<td style="width:107px;">Life Technologies</td>
			<td style="width:119px;">15557-036</td>
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			<td height="42" style="height:42px;width:216px;">Non-fat dry milk</td>
			<td style="width:107px;">Lab Scientific Inc</td>
			<td style="width:119px;">M0841</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ECL</td>
			<td style="width:107px;">Thermo Fisher</td>
			<td style="width:119px;">80196</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">X-ray film</td>
			<td style="width:107px;">Genesee Sci</td>
			<td style="width:119px;">30-101</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Developer</td>
			<td style="width:107px;">Konica Minolta</td>
			<td style="width:119px;">SRX-101A</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">UV Crosslinker</td>
			<td style="width:107px;">Stratagene</td>
			<td style="width:119px;">XLE-1000</td>
			<td></td>
		</tr>
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			<td height="84" style="height:84px;width:216px;">Sonicator</td>
			<td style="width:107px;">Branson Sonifier&#160; Digital Ultrasonic Cell Disruptor</td>
			<td style="width:119px;">Model: 450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:119px;">Model: 5810R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Water bath</td>
			<td style="width:107px;">Fisher Scientific Isotemp</td>
			<td style="width:119px;">Model: 2322</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoDrop 1000 spectrophotometer</td>
			<td style="width:107px;">ThermoScientific</td>
			<td style="width:119px;">Model: 1000</td>
			<td></td>
		</tr>
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			<td height="63" style="height:63px;width:216px;">Slot Blot apparatus&#160;</td>
			<td style="width:107px;">Schleicher and Schuell Minifold II</td>
			<td style="width:119px;">44-27570</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Tissue Culture Incubator</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:119px;">Series II 3110 Water-Jacketed CO2 Incubators</td>
			<td></td>
		</tr>
	</tbody>
</table>

examination of proteins bound to nascent dna in mammalian cells using  brdu-chip-slot-western technique

组蛋白脱乙酰1和2（HDAC1,2）定位于DNA复制的位点。在以前的研究中，使用的选择性抑制剂和遗传击倒系统，我们在复制叉进展和在哺乳动物细胞中染色质新生维护表明新颖功能HDAC1,2。此外，我们使用了结合的溴脱氧尿苷（尿嘧啶）标记的DNA染色质免疫沉淀（ChIP）与狭槽印迹和西方分析定量测量与新生的DNA结合蛋白或组蛋白修饰的BrdU芯片插槽西方的技术。 活跃分裂细胞用HDAC1,2选择性抑制剂或转染的抗HDAC1和 HDAC2的siRNA，然后新合成的DNA进行标记与胸苷类似物溴脱氧尿苷（BrdU）标记。 BrdU标记做在一个时间点没有显著细胞周期阻滞或凋亡因HDAC1,2功能的丧失。用L细胞用BrdU的组蛋白乙酰化的痕迹，染色质免疫沉淀或染色质remodeler的abeling用特异性抗体进行。 BrdU标记输入DNA和免疫沉淀（或ChIPed）DNA随后点样到使用狭缝印迹技术的膜和使用UV固化。新生DNA的每个时隙的数量，然后定量地使用Western分析用抗BrdU抗体进行评估。对新合成的DNA相关的组蛋白乙酰化的标记和染色质remodeler水平HDAC1,2功能损耗的效果然后通过从处理过的样品，以对照样品中获得的BrdU的芯片信号标准化来确定。

为了确定HDAC1,2选择性抑制剂的特异性，Hdac1,2 FL / FL和 HDAC3 FL / FL纤维肉瘤细胞。 Cre重组酶（AD-CRE）含有腺病毒是用来删除Hdac1,2和HDAC3在这些细胞中。以下广告Cre重组感染Hdac1,2 FL / FL的 细胞中，强劲增长H4K5ac进行了观察。 Hdac1,2敲除细胞用233或898的治疗不导致确认233和898抑制Hdac1,2和不HDAC3在这?...

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Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

Research

JoVE Journal

Biology

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

游离成年哺乳动物元DNA的细胞核内内微量注射

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

使用可视化的DNA复制DNA纤维的技术，在脊椎动物的模型系统DT40

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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

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 ...

Measurement of Heme Synthesis Levels in Mammalian Cells

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

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

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 ...