Name: chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae
Uploaded: 2017-12-29T15:00:00.000+00:00
Duration: 11 min 6 s
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。 注: 对于许多组蛋白 PTM 抗体, 每 IP 1-3 µg 抗体就足够了。然而, 建议的滴定实验, 以确定适当的浓度。推荐浓度的良好, 商业组蛋白 PTM 抗体往往是准确的, 可以在发表的报告或产品文献中找到。</li>
	<li>将珠子放在磁性的支架上, 除去上清液。用1毫升的 TBS 洗涤。</li>
	<li>重在20µL 的 tbs 每 IP 样品加一个额外的5µL (万一移错误) 的 tbs。 注: 珠子可在4° c 的短期贮存, 直到使用。</li>
</ol>
2. 培养酵母细胞
<ol>
	<li>接种10毫升的 YPD (10% 酵母提取物, 20% 蛋白胨和20% 葡萄糖) 与适当的菌株的单一菌落和生长在30° c 隔夜在震动孵化器在 220 rpm。</li>
	<li>用分光光度计测量酵母培养物 600 nm (OD600) 的光学密度。将区域性稀释到 OD600 = 0.2 在100毫升 YPD 中的新烧瓶中。</li>
	<li>在30° c 的振动孵化室中, 在 220 rpm 处生长细胞, 直至达到 mid-log 阶段 (OD600 = 0.6-0.8)。</li>
</ol>
3.体内蛋白质与 DNA 的交联
<ol>
	<li>当培养物达到 mid-log 阶段时, 将2.7 毫升的37% 甲醛直接添加到培养基中, 最终浓度为1% 的甲醛。将烧瓶转到25° c (或室温) 的振动器, 并在 50 rpm 处摇动15分钟。</li>
	<li>将5毫升的2.5 米甘氨酸添加到培养基中, 在室温下连续晃动50分钟, 使甲醛淬火5分钟。</li>
	<li>转移细胞到离心瓶和旋转细胞在 5800 x g 为5分钟在4° c。醒酒上清。
	<ol>
		<li>通过溶液45毫升的冷 TBS 将细胞洗净, 并将悬浮液转化为50毫升的锥形管。旋转管在 2800 x g 为3分钟在4° c。除去上清液。</li>
		<li>在10毫升的冷芯片裂解缓冲液中洗涤细胞 (50 毫米三盐酸 pH 7.5, 150 毫米氯化钠, 1 毫米乙酸酸 (EDTA), 1% nonidet 辛基 phenoxypolyethoxylethanol (NP-40), 贮存在4° c)。</li>
		<li>旋转细胞在 2800 x g 为3分钟在4° c。除去上清液。 注: 细胞可以用液氮冷冻, 贮存在-80 ° c, 直至进一步加工。</li>
	</ol>
	</li>
</ol>
4. 制作酵母裂解
<ol>
	<li>重1毫升的冷芯片裂解缓冲液中的1毫米磺氟化物 (PMSF) 和 1:1, 000 稀释酵母蛋白酶抑制剂鸡尾酒。将悬浮液转移至2.0 毫升的螺旋帽管, 内含200µL 的玻璃珠。</li>
	<li>将电池转移到4° c 的高速搅拌的微珠跳动装置, 每三十年代6次。保持在冰上的细胞至少1分钟之间的每个珠殴打。</li>
	<li>使用凝胶加载技巧将裂解液转化为1.5 毫升的管子。离心裂解液在 15500 x g 为20分钟在4° c。</li>
	<li>丢弃上清和重250µL 的 MNase 消化缓冲剂 (10 毫米三盐酸 pH 7.5, 10 毫米氯化钠, 3 毫米氯化镁2, 21 mm CaCl2, 1% NP-40, 存储在4° c) 轻轻移向上和向下。</li>
	<li>在反应中加入2.5 µL 的 MNase。将它们轻轻地搅拌4-6 次, 并立即在37° c 水浴中孵育20分钟。 注意: 当使用新的 MNase 时, 应优化消化条件。请参见协议的步骤10。</li>
	<li>立即放置在冰上的管停止反应, 并增加5µL 0.5 米 edta 最后浓度10毫米 edta。用倒装法轻轻地混合。</li>
	<li>离心反应在 15500 x g 为15分钟在4° c 和转移上清到一个新的1.5 毫升管子。 注: 可溶性 (消化) 染色质将在上清。颗粒含有任何未消化和不溶的细胞碎片。</li>
</ol>
5. Immunoprecipitate (IP) 修饰组蛋白
<ol>
	<li>使用布拉德福德法测定每 MNase 释放的染色质分数的蛋白质浓度。添加1毫升布拉德福德试剂每8一次性试管加1额外试管的每一个蛋白质样品要测试。
	<ol>
		<li>准备一个2毫克/毫升牛血清白蛋白 (BSA) 的库存溶液。</li>
		<li>添加适当的体积以达到以下浓度的 BSA 在1毫升布拉德福德试剂: 0 微克/毫升, 0.5 微克/毫升, 1 微克/毫升, 2 微克/毫升, 4 微克/毫升, 6 微克/毫升, 8 微克/毫升和10微克/毫升。添加2μ l MNase 释放的染色质分数的额外的试管。</li>
		<li>覆盖每个试管的顶部与一小块膜和反转的试管4-6 倍, 以混合解决方案好。室温下孵育试管15分钟。</li>
		<li>使用可见光分光光度计测量标准曲线和实验样品的 595 nm 的吸光度。 注: 在进行第一次测量之前, 使用无 BSA (0 微克/毫升) 的试管空白分光光度计。</li>
		<li>使用与分光光度计或电子表格软件相关的软件的不同浓度的 BSA 的吸光度值绘制标准曲线。根据标准曲线和所使用的稀释因子 (1:500), 使用吸光度值计算每个染色质分数样品的蛋白质浓度。</li>
	</ol>
	</li>
	<li>对于每个 IP, 添加50µg 的总蛋白从 MNase 释放染色质分数到芯片裂解缓冲, 达到最后的体积1毫升。 注: 如果每个裂解装置设置多个 ip, 则将裂解剂和芯片裂解缓冲液的主混合, 并分1毫升用于 ip 的单个管。</li>
	<li>从 IP 混合中删除100µL 作为输入示例进行处理。将输入样本冻结在-20 ° c 直到步骤7.1。 注: 任何剩余的染色质样品也可在-20 ° c 冻结, 如果需要, 可用于后续的 IP。</li>
	<li>添加20µL 磁性蛋白 A/G 珠 pre-bound 与抗体的每个 IP 样本。</li></ol>将样品旋转 8 rpm, 3 h (标准) 或隔夜 (如果首选) 在4° c。

6. 洗涤 IPs 和洗组蛋白-DNA 复合物
<ol>
	<li>把管子放在磁性的支架上, 让珠子在管子的侧面收集。用吸管取出上清液。在下面的每个缓冲器上依次洗涤1毫升的珠子。
	<ol>
		<li>在4° c 的5分钟旋转 8 rpm, 将珠子放在磁性支架上, 除去上清, 并加入下一个缓冲器: 2x-片状裂解缓冲液, 2x-高盐洗涤缓冲器 (50 毫米三盐酸 pH 7.5, 500 毫米氯化钠, 1 毫米 EDTA, 1% NP-40, 储存在4° c), 1x-氯化/洗涤剂洗涤缓冲 (10 毫米三盐酸盐 pH 8.0, 250 毫米氯化, 1 毫米 edta, 1% NP-40, 1% 钠胆酸, 存储在4° c), 1x te (10 毫米三盐酸 pH 8.0, 1 毫米 edta, 存储在4° c), 和 1x te。</li>
		<li>用最后的 te 洗涤, 转移珠子到一个新的管使用0.5 毫升 te 转移大部分的珠子, 然后再0.5 毫升的 te 清洗管和转移剩余的珠子。</li>
	</ol>
	</li>
	<li>添加250µL 新制作的芯片洗脱缓冲器 (1% SDS, 1 mM NaHCO3)。简要地漩涡解答。在室温下旋转 8 rpm 的样品15分钟。</li>
	<li>把管子放在磁性的支架上, 收集珠子。小心地将上清液转移到新的管子上, 避免珠子。 注: 洗含有 immunoprecipitated 蛋白和相关的 DNA。</li>
	<li>再添加250µL 的芯片洗脱缓冲珠。在室温下旋转 8 rpm 的样品15分钟。</li>
	<li>小心地将上清液转移到含有第一洗脱的管子。如有必要, 卸下所有 IPs 的较小音量 (240 µL) 以避免干扰珠子。</li>
</ol>
7. 反向蛋白质-DNA 通道
<ol>
	<li>解冻输入样本, 并在每个输入中添加400µL 的芯片洗脱缓冲器。</li>
	<li>对输入和 IP 样本, 添加20µL 5 米氯化钠, 5 µL 20 毫克/毫升糖原, 12.5 毫克/毫升蛋白酶 k µL 的混合好, 通过倒置或弹管。在一夜间65° c 水浴中孵育样品。</li>
</ol>
8. 纯化和浓缩 DNA
<ol>
	<li>添加10µL 10 毫克/毫升核糖核酸 A 到输入和 IP 样本。在37° c 水浴中孵育样品30分钟。</li>
	<li>在水溶液中加入600µL phenol:chlorofrom:isoamyl 醇 (25:24:1 PCI), 并将它们混合均匀。在室温下旋转 15500 x g 5 分钟。
	<ol>
		<li>将水层移至新管。加入600µL 的 PCI, 并将它们混合均匀。在室温下旋转 15500 x g 的管子5分钟。将水层转移到新的管中。 警告: 在使用 PCI 时, 应在通风橱中工作并佩戴适当的个人防护设备。按照机构指导方针的指示处理液体和固体废物。</li>
	</ol>
	</li>
	<li>添加0.1x 体积的3M 醋酸钠和1毫升的冷100% 乙醇沉淀的 DNA。倒置管4-6 次, 以混合解决方案好。在晚上-20 ° c 孵育。
	<ol>
		<li>旋转管在 15500 x g 为20分钟在4° c。小心地用吸管把上清液取出。</li>
		<li>用1毫升的冷70% 乙醇清洗颗粒。旋转它在 15500 x g 为10分钟在4° c。小心地用吸管把上清液取出。</li>
		<li>空气烘干药丸在敞篷为大约20分钟 (直到完全地干燥)。重 IP 样品在50µL 核酸水和重输入样品在100µL 无核酸水。将 DNA 样本储存在-20 ° c 直到执行 qPCR。</li>
	</ol>
	</li>
</ol>
9. 定量 PCR (qPCR) 检测富集基因组区域
<ol>
	<li>为每一组底漆制作一个 qPCR 的主组合。一个反应包含5µL 的 2x qPCR 混合, 0.25 µL 20 µM 前引物, 和0.25 µL 20 µM 反向底漆。 注: qPCR 实验的适当入门设计和测试在其他地方描述14,15,16。</li>
	<li>为每个 dna 样本制作 dna 主混合。一个反应包含0.5 µL 的 DNA 和4.0 µL 的核酸水。</li>
	<li>添加5.5 µL 适当的底漆主混合和4.5 µL 适当的 DNA 主混合每井在一个384井 qPCR 板。 注: 开始添加5.5 µL 的底漆混合到每一个井。在室温下在 200 x g 旋转1分钟, 然后再加入4.5 µL 的 DNA 混合。</li>
	<li>在室温下, 将密封粘附到板上, 在 200 x g 处旋转1分钟。</li>
	<li>使用具有以下条件的 real-time qPCR 系统执行 qPCR:95 ° c 为3分钟;40周期95° c 为十年代, 55 ° c 为三十年代;熔化曲线65° c 到95° c 以0.5 ° c 增量, 5 s。</li>
	<li>对于每个入门对, 计算 IP 样本相对于 qPCR 中使用的5% 输入样本的百分比输入。利用技术 PCR triplicates 的方法进行计算。 注意: 如果在 PCR triplicates 中观察到大量的变异, 最好是在384孔板中, qPCR 注意主成分组合、移误差和井间交叉污染。<ol>
		<li>通过从原始的值中减去代表稀释因子的周期数, 将输入的值调整为100%。 注: 5% 输入表示20倍的稀释因子, 等于4.32 循环 (日志2 20 = 4.32)。</li>
		<li>将输入百分比计算为 2 ^ (重庆输入-重庆IP) 乘以100。</li>
	</ol>
	</li>
</ol>
10. 确定 MNase 消化条件 (在第一次全芯片试验之前推荐)
<ol>
	<li>按照上述协议的步骤2.1 到4.4。扩大协议, 以产生足够的裂解物的多个样品的 MNase 消化的染色质含有颗粒。在步骤 4.4, 重在1.25 毫升的 MNase 消化缓冲颗粒。将样品分到5根管子, 使每一个都含有250µL 的染色质颗粒悬浮在 MNase 消化缓冲液中。</li>
	<li>每管加0、1.5、2.5 或5µL MNase。将它们轻轻地搅拌4-6 次, 然后立即在37° c 水浴中孵育20分钟的管子。</li>
	<li>停止反应, 立即放置在冰上的管和增加5µL 0.5 米 EDTA 为10毫米最终浓度。</li></ol>通过反转将溶液轻轻混合。
	<li>离心管在 15500 x g 为15分钟在4° c 和转移上清到一个新的1.5 毫升管子。</li>
	<li>添加 SDS 到1% 最终浓度 (25 µL 10% 库存溶液) 和 NaHCO3到0.1 米最终浓度 (25 µL 1M 库存溶液) 的样品在1.5 毫升管。</li>
	<li>添加20µL 5M 氯化钠, 5 µL 糖原, 和12.5 µL 20 毫克/毫升蛋白酶 K 的样品在1.5mL 管。在晚上65° c 孵育。</li>
	<li>添加10µL 10 毫克/毫升 RNaseA。孵育在37° c 为30分钟。</li>
	<li>在水溶液中加入300µL 的 PCI, 并将其混合均匀。在室温下旋转 15500 x g 5 分钟。
	<ol>
		<li>将水层移至新管。添加300µL 的 PCI 和混合好。在室温下旋转 15500 x g 5 分钟。将水层转移到新的管中。</li>
	</ol>
	</li>
	<li>添加0.1x 的3米醋酸钠 (通常为30µL) 和1毫升的冷100% 乙醇沉淀的 DNA。倒置管4-6 次混合好。在-80 ° c 下孵育1小时或-20 ° c 过夜。
	<ol>
		<li>旋转管在 15500 x g 20 分钟, 在4° c。小心地用吸管除去上清液。</li>
		<li>在1毫升的冷70% 乙醇中清洗颗粒。旋转在 15500 x g 为10分钟在4° c。</li>
		<li>让小球在引擎盖风干大约20分钟 (或直到完全地干燥)。重30µL 的核酸水中的小球。</li>
	</ol>
	</li>
	<li>添加 dna 加载染料, 并运行20µL 在一个2% 琼脂糖凝胶可视化消化 dna。</li>

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(86), (2014).</li><li>Kuo, M. H., 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=In+vivo+cross-linking+and+immunoprecipitation+for+studying+dynamic+Protein:DNA+associations+in+a+chromatin+environment.">In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment.</a> Methods. 19 (3), 425-433 (1999).</li><li>Meluh, P. B., Broach, J. 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B., 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=An+Interactive+Database+for+the+Assessment+of+Histone+Antibody+Specificity.">An Interactive Database for the Assessment of Histone Antibody Specificity.</a> Mol Cell. 59 (3), 502-511 (2015).</li></ol>

作者没有什么可透露的。

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><tbody><tr><td>Yeast Extract</td><td>Research Products International (RPI)</td><td>Y20025-1000.0</td><td></td></tr><tr><td>Peptone</td><td>Research Products International (RPI)</td><td>P20250-1000.0</td><td></td></tr><tr><td>Dextrose</td><td>ThermoScientific</td><td>BP350-1</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>AC12007-0050</td><td></td></tr><tr><td>Tris</td><td>Amresco</td><td>0497-5KG</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758-500G</td><td></td></tr><tr><td>NaCl</td><td>ThermoScientific</td><td>BP358-10</td><td></td></tr><tr><td>4-Nonylphenyl-polyethylene glycol</td><td>Sigma-Aldrich</td><td>74385</td><td>Equivalent to NP-40</td></tr><tr><td>MgCl</td><td>ThermoScientific</td><td>S25533</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>20899-25G-F</td><td></td></tr><tr><td>LiCl</td><td>ThermoScientific</td><td>AC413271000</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Amresco</td><td>M107-1KG</td><td></td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>30970-100G</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>ThermoScientific</td><td>S25533</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626-5G</td><td></td></tr><tr><td>Yeast protease inhibitor cocktail</td><td>VWR</td><td>10190-076</td><td></td></tr><tr><td>25 Phenol:24 Chloroform:1 Isoamyl Alcohol</td><td>VWR Life Science</td><td>97064-824</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Nuclease-Free Water</td><td>VWR</td><td>100720-992</td><td></td></tr><tr><td>Micrococcal Nuclease</td><td>Worthington Biochemical</td><td>LS004797</td><td></td></tr><tr><td>Glycogen</td><td>ThermoScientific</td><td>R0561</td><td></td></tr><tr><td>Proteinase K</td><td>Research Products International (RPI)</td><td>P50220-0.1</td><td></td></tr><tr><td>RNase A&amp;nbsp;</td><td>Sigma-Aldrich</td><td>R6513-50MG</td><td></td></tr><tr><td>&amp;nbsp;Bradford Assay Reagent</td><td>ThermoScientific</td><td>23238</td><td></td></tr><tr><td>&amp;nbsp;BSA Standard 2 mg/mL</td><td>ThermoScientific</td><td>23210</td><td></td></tr><tr><td>&amp;alpha; H4</td><td>EMD Millipore</td><td>04-858</td><td></td></tr><tr><td>&amp;alpha; H4K16ac</td><td>EMD Millipore</td><td>ABE532</td><td></td></tr><tr><td>&amp;alpha; H3</td><td>Abcam</td><td>ab1791</td><td></td></tr><tr><td>&amp;alpha; H3K4me2</td><td>Active Motif</td><td>39142</td><td></td></tr><tr><td>&amp;nbsp;High Rox qPCR Mix</td><td>Accuris qMax Green, Low Rox qPCR Mix</td><td>ACC-PR2000-L-1000</td><td></td></tr><tr><td>Protein A/G Magnetic Beads</td><td>ThermoScientific</td><td>88803</td><td></td></tr><tr><td>magnetic stand for 1.5mL tubes</td><td>Fisher Scientific</td><td>PI-21359</td><td></td></tr><tr><td>Acid-Washed Glass Beads</td><td>Sigma-Aldrich</td><td>G8772</td><td></td></tr><tr><td>&amp;nbsp;Microtube Homogenizer</td><td>Benchmark</td><td>D1030</td><td></td></tr><tr><td>2.0 mL screw-cap tubes with sealing rings</td><td>Sigma-Aldrich</td><td>Z763837-1000EA</td><td></td></tr><tr><td>Gel loading tips</td><td>Fisher Scientific</td><td>07-200-288</td><td></td></tr><tr><td>Cuvettes</td><td>Fisher Scientific</td><td>50-476-476</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-10</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>384-Well PCR Plate</td><td>Fisher Scientific</td><td>AB-1384W</td><td></td></tr><tr><td>&amp;nbsp;Gyratory Floor Shaker</td><td>New Brunswick Scientific</td><td>Model G10</td><td></td></tr><tr><td>Spectrophotometer</td><td>ThermoScientific</td><td>ND-2000c</td><td></td></tr><tr><td>&amp;nbsp;Real-Time PCR Detection System</td><td>Bio-Rad</td><td>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 ...

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Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Unit 1

DNA and Chromosome Structure

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12.6K 次观看.  University of Maryland. 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。

视频: Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

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

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

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

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

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 ...

科研

遗传学

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

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken 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, ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

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

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

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

Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

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 ...

从酵母细胞中分离基因座特异性染色质

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

作者聚焦：使用高富集位点特异性染色质分离方案推进染色质研究并克服限制 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

酿酒酵母的单拷贝基因位点染色质纯化

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

生物化学

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

神经科学

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

生物学

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

从酿酒酵母中组蛋白修饰的染色质沉淀 (芯片)

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与ChEC-seq的蛋白质-DNA相互作用的全基因组映射

低丰度胚胎标本的染色质沉淀 (芯片) 协议

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

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

酿酒酵母的单拷贝基因位点染色质纯化

酿酒酵母的单拷贝基因位点染色质纯化

酿酒酵母的单拷贝基因位点染色质纯化

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景

蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的