Name: chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae
Uploaded: 2017-12-29T15:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

作者想感谢格林实验室的成员进行有益的讨论。这项工作得到了部分由 NIH 赠款 R03AG052018 和 R01GM124342 E.M.G。

1. 磁珠预绑定抗体
<ol>
	<li>混合蛋白 A/G 磁性珠井和转移20µL 磁珠每一个沉淀 (IP) 样品到1.5 毫升管。 注: 当移珠, 使用宽口径, low-retention 提示。</li>
	<li>把管子与珠子放在一个磁性的立场, 并允许珠子收集在一侧的管。除去上清液。</li>
	<li>用1毫升的冷三缓冲盐水 (TBS) 洗涤珠子3次 (50 毫米三盐酸 pH 7.5, 150 毫米氯化钠)。</li>
	<li>添加200µL 的 TBS 到珠子和适当数量的抗体。在4° c 的夜间旋转 8 rpm。...

<ol>
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这里描述的过程允许有效地恢复 DNA 与修饰组蛋白相关的酵母细胞中的沉淀。随后, qPCR 使用引物放大感兴趣的区域, 以确定局部富集或特定组蛋白修饰的损耗。尽管在近20年前被开发成为一种方法, 但芯片仍然是研究组蛋白修饰状态在不同的染色体区域和在各种条件下的决定性的检测。尽管芯片耦合到下一代测序技术可以在全基因组范围内对蛋白修饰进行审问6,<sup class="xre...

组蛋白的动态修饰修饰 (PTM) 是许多 dna 模板化过程的关键调控机制, 包括转录、复制和 dna 修复1,2。因此, 确定修饰组蛋白的丰度和精确定位与这些过程相伴的能力, 对于了解它们在细胞不同条件下的调节是至关重要的。染色质沉淀 (芯片) 的发展主要来源于生物化学研究的蛋白质与 DNA 的相互作用, 特别是体外方法使用化学交, 再加上需要评估蛋白质-DNA 相互作用的动态性质在体内和在基因组的特定区域3,4,5。定量 PCR (qPCR) 和测序技术的进步也扩大了进行芯片实验的能力, 以定量比较和整个基因组, 使之成为解剖 DNA-蛋白质相互作用的有力工具多个级别。
目前, 芯片是一个必要的方法, 任何研究组感兴趣的染色质介导的基因组的调节, 因为有没有可比的方法直接询问的物理联系的修饰组蛋白和特定的基因座的轨迹在体内。尽管此方法的变体使用下一代测序来映射组蛋白修饰, 在整个染色体6,7中可用, 但这些方法可以解决不同的科学问题及其规模、成本和技术资源可能限制一些研究组。此外, 有针对性的芯片 qPCR 是必要的, 以补充这些方法, 提供的方式, 以优化的芯片协议之前, 排序和验证的结果, 从表数据集。质谱法的方法, 以确定完整补充的组蛋白标记相关的基因区域也出现了8,9,10,11, 但是, 这些方法有一些限制, 关于哪些区域的基因组可以被探测, 他们需要的技术专长和仪器, 将无法提供给所有研究组。因此, 芯片仍然是分析组蛋白修饰在不同条件下的丰度和分布的基础方法, 对所有感兴趣的研究小组的遗传学, 染色质和基因组功能的调节。
在这里, 我们描述了一个方法的芯片使用萌芽酵母模型酿酒酵母 (S.) , 以研究组蛋白 PTMs 在染色质上的分布。这种方法依赖于在酵母中开发的一些芯片协议的核心组件, 也适用于不同的模型系统12,13。修饰组蛋白与 DNA 在细胞内的相互作用与甲醛交联保存。在裂解制备后, 染色质碎片通过消化 micrococcal 核酸溶解成 uniformly-sized 片段。修饰组蛋白的沉淀是用商业或实验室生成的抗体进行的, 任何相关的 DNA 都是用 qPCR (图 1) 分离和分析的, 以便在特定的染色体区域富集。对于许多组蛋白修饰, 从这个协议获得的 DNA 的数量是足够的测试超过25不同的基因座的 qPCR。
这种芯片方法是高度通用的监测在多个突变株或环境条件下的单一组蛋白修饰的分布, 或测试多组蛋白修饰在野生细胞的多个基因座基因座。此外, 该协议的许多组成部分很容易调整, 以优化检测无论是高或低丰富的组蛋白标记。最后, 在芽酵母中修饰组蛋白的执行芯片提供了使用关键的控制抗体特异性的机会, 在其他系统中基本上是不可用的。即, 酵母菌株可以产生, 携带点突变的组蛋白残留的目标是修改, 在某些情况下, 只有一个单一的酶, 催化修饰特定的组蛋白残留 (e. g. 组蛋白赖氨酸甲基)。因此, 芯片可以在组蛋白突变体或酶缺失菌株中进行, 以测定抗体的非特异性结合程度, 并产生假阳性结果。这种控制是特别有价值的新发展的抗体, 甚至可以用来验证抗体特异性的保守组蛋白修饰之前, 其使用的其他系统。这种方法补充了其他方法来测试抗体特异性, 区分不同的修饰状态 (如单, di 和三甲基化), 包括探针阵列的修饰肽和执行西方印迹的组蛋白或核具有定义的修改。总的来说, 芽酵母的芯片是一种强有力的方法来评估组蛋白 PTMs 的动态, 在整个染色体和解剖的机制控制他们的调节。

<table border="0" cellpadding="0" cellspacing="0" width="876">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast Extract</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">Y20025-1000.0</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Peptone</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">P20250-1000.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Dextrose</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP350-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Glycine</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>AC12007-0050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Tris</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">0497-5KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">NaCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP358-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Nonylphenyl-polyethylene glycol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">74385</td>
			<td>Equivalent to NP-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">MgCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">CaCl2</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">20899-25G-F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">LiCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">AC413271000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Dodecyl Sulfate</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">M107-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Deoxycholate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">30970-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Acetate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">S2889</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">NaHCO3</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast protease inhibitor cocktail</td>
			<td>VWR</td>
			<td>10190-076</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">25 Phenol:24 Chloroform:1 Isoamyl Alcohol</td>
			<td style="width:301px;">VWR Life Science</td>
			<td>97064-824</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ethanol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Nuclease-Free Water</td>
			<td style="width:301px;">VWR</td>
			<td style="width:164px;">100720-992</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Micrococcal Nuclease</td>
			<td>Worthington Biochemical</td>
			<td>LS004797</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Glycogen</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">R0561</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Proteinase K</td>
			<td>Research Products International (RPI)</td>
			<td>P50220-0.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">RNase A&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>R6513-50MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Bradford Assay Reagent</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">23238</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;BSA Standard 2 mg/mL</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>23210</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H4</td>
			<td>EMD Millipore</td>
			<td>04-858</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H4K16ac</td>
			<td>EMD Millipore</td>
			<td>ABE532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3</td>
			<td style="width:301px;">Abcam</td>
			<td style="width:164px;">ab1791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3K4me2</td>
			<td>Active Motif</td>
			<td>39142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;High Rox qPCR Mix</td>
			<td>Accuris qMax Green, Low Rox qPCR Mix</td>
			<td>ACC-PR2000-L-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Protein A/G Magnetic Beads</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>88803</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">magnetic stand for 1.5mL tubes</td>
			<td>Fisher Scientific</td>
			<td>PI-21359</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Acid-Washed Glass Beads</td>
			<td>Sigma-Aldrich</td>
			<td style="width:164px;">G8772</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Microtube Homogenizer</td>
			<td style="width:301px;">Benchmark</td>
			<td>D1030</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">2.0 mL screw-cap tubes with sealing rings</td>
			<td>Sigma-Aldrich</td>
			<td>Z763837-1000EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Gel loading tips</td>
			<td>Fisher Scientific</td>
			<td>07-200-288</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Cuvettes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">50-476-476</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Parafilm</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">50 mL conical tubes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">384-Well PCR Plate</td>
			<td>Fisher Scientific</td>
			<td>AB-1384W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Gyratory Floor Shaker</td>
			<td style="width:301px;">New Brunswick Scientific</td>
			<td style="width:164px;">Model G10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Spectrophotometer</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">ND-2000c</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">&#160;Real-Time PCR Detection System</td>
			<td style="width:301px;">Bio-Rad</td>
			<td style="width:164px;">1855485</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae

组蛋白修饰修饰 (PTMs), 如乙酰化, 甲基和磷酸, 是由一系列酶动态调节的, 它们可以添加或除去这些标记, 以响应细胞接收到的信号。这些 PTMS 是调控基因表达调控和 DNA 修复等过程的关键因素。染色质沉淀 (芯片) 一直是一个工具的方法, 以解剖的丰度和本地化的许多组蛋白 PTMs 整个染色体, 以响应对细胞的各种扰动。在这里, 一个多功能的方法来执行芯片的 post-translationally 修饰组蛋白从萌芽酵母酿酒酵母 (S. 酵母)描述。该方法依赖于用甲醛处理酵母培养物的蛋白质和 dna 的交联, 通过微珠打浆产生酵母裂解, micrococcal 核酸和组蛋白 DNA 的沉淀对染色质片段的增溶。配合.与组蛋白标记相关的 DNA 被纯化并进行定量 PCR 分析, 以评估其在多个基因座上的富集。对野生和突变酵母中组蛋白标记 H3K4me2 和 H4K16ac 定位的代表性实验进行了探讨, 以验证数据分析和解释。该方法适用于多种组蛋白 PTMs, 可与不同的突变菌株或在不同环境应力的情况下进行, 使其成为研究各种条件下染色质动力学变化的极好工具。

该协议的一个关键组成部分是优化 micrococcal 核酸 (MNase) 的浓度, 用于消化染色质到可溶性片段, 如步骤10所述。这对于获得关于组蛋白修饰在感兴趣的染色体区域分布的高分辨率数据至关重要。应进行 MNase 滴定, 以确定最合适的浓度, 以实现主要单核与少量的 di 核在可溶性染色质分数。这可以通过提取的 DNA 后 MNase 消化的染色质含有颗粒和执行琼脂糖凝胶电泳 (<strong class="x...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

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)

在这里, 我们描述了一个协议的染色质沉淀的修饰组蛋白从萌芽酵母酿酒酵母。Immunoprecipitated DNA 随后用于定量 PCR 研究组蛋白修饰修饰的丰度和局部化。

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

The overall goal of this chromatin immunoprecipation protocol is to determine the abundance and localization of histone modifications at defined regions throughout the budding yeast genome. This method can help answer key questions in chromatin regulation, such as how histone modifications are linked to gene expression control and how these modifications change in response to different environmental conditions. An extremely versatile protocol that can be used to investigate many different histone modifications and multiple E strains at one time.Though this method can provide insight into histone modifications, it can also be applied to investigating other chromatin bonding proteins through the use of epitope tags or available antibodies. Start this procedure by growing the yeast cells inoculate 10 milliliters of YPD medium with a single colony of each appropriate strain, and grow it 30 degrees celsius in a shaking incubator at 220 rpm overnight. On the following day after measuring the optical density at 600 nanometers or OD600 of the culture, dilute the culture to an OD600 of 0.2 in 100 mL of YPD in a new flask.Grow the cells at 30 degree celsius in a shaking incubator at 220 rpm until they reach mid log phase. When the cultures have reached mid log phase, add 2.7 milliliters of 37%formaldehyde directly to the medium in each flask for a final concentration of 1%formaldehyde. Transfer the flasks to a shaker at room temperature, and shake at 50 rpm for 15 minutes.Add five milliliters of 2.5 molar glycine to the medium and continue shaking at 50 rpm at room temperature for five minutes to quench the formaldehyde. Subsequently, spin down the cells and wash them as described in the text protocol. Remove the supernatant after the last spin and proceed to making yeast lysates.Re-suspend the cells in one milliliter of cold chromatin amino precipitation or ChIP Lysis buffer with 1 millimolar PMSF and 1:1000 dilution of a yeast protease inhibitor cocktail. Transfer the suspension to a 2.0 milliliter screw cap tube containing 200 microliters of glass beads. Transfer the cells to a bead-beating apparatus for a high speed agitation at four degree celsius.Bead-beat for 30 seconds and ice for one minute. Bead-beat a total of six times, keeping the cells on ice for at least one minute between each 30 second beat-beating. Using a gel loading tip, transfer the lysate to a 1.5 milliliter tube.Centrifuge the lysate at 15, 500 g at four degree celsius for 20 minutes. Discard the supernatant and re-suspend the pellet in 250 microliters of micrococcal nuclease digestion buffer by gently pipetting up and down. Add 2.5 microliters of micrococcal nuclease to each reaction.Mix gently by inverting four to six times and immediately incubate in a 37 degree celsius water bath for 20 minutes. After 20 minutes, stop the reaction by placing the tubes on ice, adding five milliliters of 0.5 molar EDTA for a final concentration of 10 millimolar EDTA and mixing gently by inversion. Centrifuge at 15, 500 g at four degree celsius for 15 minutes.Then, transfer each supernatant to a new 1.5 milliliter tube. For the most consistent results, it is critical to ensure that the same concentration of protein is used in each amino precipitation. Depending on the number of amino precipitation or IP reactions, make a master mix of each micrococcal nuclease released chromatin fraction and ChIP lysis buffer and allocate one milliliter containing 50 micrograms of total protein to individual tubes.Remove 100 microliters from each IP mix to process as the input sample. Freeze the input samples at 20 degree celsius for later use. It is also important to make sure the same amount of beads bound to the antibody is used for each immunoprecipitation.Using a wide board pipette tip, add 20 microliters of magnetic protein AG beads, pre-bound with antibody to each IP sample. Rotate the samples at eight rpm at four degree celsius for three hours. When the IP is done, place the tubes in a magnetic stand and let beads collect on the side of the tube.Use a pipette to remove the supernatant. Add one milliliter of ChIP lysis buffer to the beads and rotate at eight rpm at four degree celsius for five minutes. Then, place the tubes on the magnetic stand and remove the supernatant.In this manner, wash the beads successively with the following buffers for the number of times indicated. ChIP Lysis Buffer, High Salt Wash Buffer, LiCl/detergent Wash Buffer and TE.With the last TE wash, use 0.5 milliliters of TE to transfer most of the beads to a new tube, and then use another 0.5 milliliters of TE to wash the tube and transfer the remaining beads. Add 250 microliters of freshly made ChIP elution buffer to each tube and vortex the solution briefly.Rotate the samples at eight rpm at room temperature for 15 minutes. Place the tubes in the magnetic stand. Carefully transfer the supernatant to a new tube and avoid the beads.Add another 250 microliters of ChIP elution buffer to the beads. Rotate the samples at eight rpm at room temperature for 15 minutes. Carefully and without disturbing the beads, transfer the supernatant to the tube containing the first elution.Thaw the input samples collected earlier and add 400 microliters of ChIP elution buffer to each input sample. To reverse protein DNA cross links, add to both input and IP samples, 20 microliters of five molar sodium chloride, five microliters of glycogen and 12.5 microliters of Proteinase K.Mix well by inverting or flicking the tubes. Incubate the samples in a 65 degree celsius water bath overnight.To begin this procedure, make a QPCR master mix for each set of primers by combining the appropriate volumes of QPCR mix, the forward primer, and the reverse primer. Make DNA master mixes for each DNA sample where one reaction contains 0.5 microliters of DNA and 4.0 microliters of nuclease free water Add 5.5 microliters of the appropriate primer master mix to each well in a 384 well QPCR plate. Spin the plate at 200 g at room temperature for one minute.Then, add 4.5 microliters of the appropriate DNA master mix to each well. Seal the plate, and spin at 200 g at room temperature for one minute. Perform QPCR using a real time QPCR system with the following conditions, 95 degree celsius for three minutes 40 cycles of 95 degree celsius for 10 seconds and 55 degree celsius for 30 seconds and a melting curve of 65 degree celsius to 95 degree celsius with 0.5 degree celsius increments for five seconds.One key component of this protocol is optimizing the concentration of micrococcal nuclease used to digest the chromatin into soluble fragments. This example result from agarose gel electrophoresis of DNA isolated following digestion of the chromatin pellet with varying amounts of micrococcal nuclease shows that 2.5 microliters of enzyme at 20 units per microliter produced predominantly mononucleosomes. This representative ChIP experiment used antibodies against a histone methyl marker H3K4me2 and histone H3 in wild type and set1 knockout cells.Set1 knockout cells lack the methyl transferase that catalyzes the mark The bars indicate the relative enrichment of H3K4me2 at the indicated low sci. The wild type strain showed clear enrichment for H3K4me2 at the five prime end of two genes known to be direct targets of set1, PMA1 and ERG11 whereas no signal was observed in set1 knockout cells. As expected, there was no enrichment of the H3K4me2 mark at TEL07L, the expression of SPR3 has also been reported to be regulated by set1 but these results indicate the regulation is likely to occur by a different mechanism than at PMA1 or ERG11.Once mastered, this technique can be done within four days if it is performed properly. While attempting this procedure, it's important to remember to carefully test the antibody used for specificity with the proper controls. If available, both positive and negative control regions should be probed in the QPCR.Following this procedure, other methods like GP sequencing can also be performed in order to determine the genome by distribution of histone modifications. After watching this video, you should have a good understanding of how to consistently detect the levels of different histone modifications at unique genomic regions with well controlled experiments.

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Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Unit 1

DNA and Chromosome Structure

SCIENTISTS IN ACTION

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

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

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 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 ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

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 ...

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 ...

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

使用麦格纳波特奥里扎伊的色度免疫沉淀测序方法对希斯通修饰分布进行全基因组分析

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...