Name: capture and identification of rna-binding proteins by using click chemistry-assisted rna-interactome capture (caric) strategy
Uploaded: 2018-10-19T14:00:00
Description: Watch this Scientific Journal Video about Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy at JoVE.com

这项工作由国家自然科学基金资助91753206、21425204和21521003和国家重点研究开发项目2016YFA0501500。

注意: 在适用的情况下, 使用的试剂应以无 rna 的形式购买, 或在无核糖核酸酶中溶解, 溶剂 (大部分为酯二乙酯 (DEPC) 处理的水)。在处理 RNA 样品和无核糖核酸酶试剂时, 务必戴上手套和口罩, 并经常更换, 以避免核糖核酸酶污染。
1. 代谢标记和 UV 交联细胞裂解物的制备
<ol><li>
欧盟和4SU 的代谢合并
	<ol>
<li>杜尔贝科的改良鹰培养基 (DMEM) 中培养的 HeLa 细胞, 辅以10% ?...

<ol>
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保持公平 RNA 完整性是成功无花果实验的关键之一。随着 Cu (I) 的适当配体和仔细操作, RNA 降解可以大大减少, 虽然部分降解被观察到。在实验样品中, 欧盟和4SU 的替代比分别为1.18% 和 0.46% (未显示数据)。对于长度为 2000 nt 的完整 rna, 约90% 的 rna 包含至少一个欧盟和一个4SU。对于部分退化的 rna, 长度为 1000 nt, 约70% 的 rna 包含至少一个欧盟和一个4SU。因此, rna 的部分降解不会显著降低无花果的效率, ?...

人类基因组转录成各种类型的编码和非编码 rna (非编码 rna), 包括 mRNAs、rRNAs、tRNAs、小核 rna (snRNAs)、小核仁 rna (snoRNAs) 和长非编码 rna (lncRNAs)1。大多数 rna 具有限制性商业惯例的服装和功能作为核糖颗粒 (RNPs)2。因此, 限制性商业惯例的综合识别是了解 rna 和限制性商业惯例之间的调控网络的先决条件, 这是涉及各种人类疾病3,4,5。
过去几年见证了限制性商业惯例在各种真核系统2,6, 包括人类7,8,9,10,11中发现的巨大的促进作用,鼠标12,13,14, 酵母9,15,16, 斑马鱼17,果蝇18,19,线虫线虫16,拟南芥20,21,22, 和人类寄生虫23,24,25.这些进步是由城堡等开发的一个限制性的捕获策略促进。7和巴尔茨等。8在 2012年, 它结合了在体内UV 交联的 RNA 及其相互作用的蛋白质, 寡核苷酸 (dT) 捕获聚 (A) rna 和质谱 (MS) 基于蛋白质组分析。然而, 鉴于聚 (a) 多数存在于成熟的 mRNAs 上, 它仅占真核转录组26的 3%-5%, 这种广泛使用的策略无法捕获与非聚 (A) rna 的限制性商业惯例交互, 包括大多数非编码 rna和预 mRNAs。
在这里, 我们报告了一个最近制定的战略的详细程序, 以转录组全捕获的聚 (a) 和非聚 (a) 限制性商业惯例27。被称为无花果, 这种策略结合体内UV 交联和 rna 代谢标记与光敏和 "可点击的" 核苷类似物 (其中包含一个叠功能组, 可以参与点击反应), 4-thiouridine (4SU) 和 5-ethynyluridine (EU)。通过无花果策略获得理想结果的关键步骤是有效的代谢标记、UV 交联和点击反应以及 RNA 完整性的维护。因为 cu (i) 作为催化剂在单击反应可能导致 rna 碎片, 一个 Cu (i) 配体, 可以减少 RNA 碎片是必不可少的。我们描述如何在细胞裂解液中执行高效的单击反应, 而不会导致严重的 RNA 降解。
虽然在 HeLa 细胞中的限制性捕获和鉴定仅在本协议中描述, 无花果策略可应用于各种细胞类型和可能的生物体。除了限制性捕获, 该协议还提供了简化的分步过程, 用于 MS 样品制备和蛋白质鉴定和定量, 这对于那些不熟悉蛋白质组实验的人是有帮助的。

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">HeLa</td>
			<td style="width:175px;">ATCC</td>
			<td style="width:175px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:351px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">11995065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">FBS (Fetal Bovine Serum)</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">10099141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Penicillin &#38; Streptomycin</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EU (5-ethynyl uridine)</td>
			<td style="width:175px;">Wuhu Huaren Co.</td>
			<td style="width:175px;">CAS:69075-42-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">4SU (4-thiouridine)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">T4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">10&#215;PBS (Phosphate-Buffered Saline)</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">AM9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">UV cross-linker</td>
			<td style="width:175px;">UVP</td>
			<td style="width:175px;">CL-1000</td>
			<td>Equiped with 365-nm UV lamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DEPC (Diethyl pyrocarbonate)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">D5758</td>
			<td>To treat water. Highly toxic!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tris&#183;HCl, pH 7.5</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">15567027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">LiCl</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">62476</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Nonidet P-40</td>
			<td style="width:175px;">Biodee</td>
			<td style="width:175px;">74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EDTA-free protease inhibitor cocktail</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">88265</td>
			<td>One tablet for 50 mL lysis buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">LDS (Lithium dodecyl sulfate)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">L9781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">15-mL ultrafiltration tube (10 kDa cutoff)</td>
			<td style="width:175px;">Millipore</td>
			<td style="width:175px;">UFC901024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">0.5-mL ultrafiltration tube (10 kDa cutoff)</td>
			<td style="width:175px;">Millipore</td>
			<td style="width:175px;">UFC501096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Streptavidin magnetic beads</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">88816</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DMSO (Dimethyl sulfoxide)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">41639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Azide-biotin</td>
			<td style="width:175px;">Click Chemistry Tools</td>
			<td style="width:175px;">AZ104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Copper(II) sulfate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">C1297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">THPTA [Tris(3-hydroxypropyltriazolylmethyl)amine]</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">762342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sodium ascorbate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">11140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Azide-Cy5</td>
			<td style="width:175px;">Click Chemistry Tools</td>
			<td style="width:175px;">AZ118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">LDS sample buffer (4&#215;)</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">NP0008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">10% bis-Tris gel</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">NP0301BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EDTA</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">AM9260G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">RNase A</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">R6513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">SDS (Sodium dodecyl sulfate)</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">15525017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NaCl</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">S3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Brij-97 [Polyoxyethylene (20) oleyl ether]</td>
			<td style="width:175px;">J&#38;K</td>
			<td style="width:175px;">315442</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Triethanolamine</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">V900257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Streptavidin agarose</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">20353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Urea</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">U5378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sarkosyl (N-Lauroylsarcosine sodium salt)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">61743</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Biotin</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">B4501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sodium deoxycholate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">30970</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">MaxQuant</td>
			<td style="width:175px;"></td>
			<td style="width:175px;"></td>
			<td>Version: 1.5.5.1</td>
		</tr>
	</tbody>
</table>

capture and identification of rna-binding proteins by using click chemistry-assisted rna-interactome capture (caric) strategy

RNA 结合蛋白 (限制性商业惯例) 的综合鉴定是了解细胞转录后调控网络的关键。一种广泛使用的策略, 用于限制性捕获利用酸化 [聚 (a)] 的靶 rna, 这主要发生在真核成熟 mRNAs, 留下大多数非聚 (A) rna 的结合蛋白不明。在这里, 我们描述了一个最近报告的方法称为单击化学辅助 RNA-路特捕获 (无花果) 的详细过程, 这使转录组范围捕获的聚 (a) 和非聚 (a) 限制性商业惯例的代谢标签结合rna,体内UV 交联, 和叠标记。

提出了质量控制步骤的代表性结果。结果包括步骤2.3.2 中所述的凝胶荧光分析的数字 (图 1)、步骤4.1.3 中描述的西方印迹分析 (图 2A) 和步骤4.2.2 中描述的银染色分析 (图 2B)。质量控制步骤对于优化无花果协议至关重要。在大规模的限制性鉴定实验的制备中始终包括质量控制。
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Watch this Scientific Journal Video about Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy at JoVE.com

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

本文介绍了应用单击化学辅助 RNA-路特捕获 (无花果) 策略来识别与编码和非编码 rna 结合的蛋白质的详细协议。

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

这种方法，CARIC，通过提供RNA结合蛋白的全面普查，可以帮助回答基因序列后转录领域的关键问题。CARIC 的主要优点是能够捕获与各种RNA结合的蛋白质，如mRNA和非编码RNA。该方法可用于各种类型和生物体的研究RNA蛋白相互作用的工作。首先，在37摄氏度的二氧化碳大气中，在37摄氏度的辅以DMEM中培养 HeLa 细胞。当细胞达到约80%的汇合时，用15毫升的预战新鲜培养剂取代每道菜上的培养培养。在每个菜中加入15微升100毫摩尔，最终浓度为1毫摩尔。对于实验和没有紫外线控制样品，每盘添加 7.5 微升 100 毫摩尔 4 SU。用铝箔盖住盘子，使用先前的条件培养细胞16小时。在此之后，将先前数量的欧盟和/或四个 SU 的一半添加到相应的板中，然后继续培养两个小时。要开始体内交叉链接，请去除培养培养剂，每次洗涤使用五毫升 PBS 用 PBS 洗三次 PBS。尽可能去除残留的 PBS。对于实验和没有四个SU控制样品，将盘子放在冰上，盖上盖子。使用 UV 交链接器在 365 纳米和 2 个百分厘米平方下用紫外光照射细胞。对于无紫外线控制样品，请将盘子放在冰上，并保护它们免受光线照射。接下来，向每个菜中加入一毫升的预解缓冲液。使用橡胶细胞提升器刮擦细胞，并在 15 毫升管中收集预解悬浮液。将预解缓冲液添加到含有从两个盘子的悬浮液的管子中，将总体积调整到六毫升。然后添加相等体积的 R 解解缓冲液。用窄针通过注射器多次通过细胞的细胞，直到酸盐清晰均匀。在四摄氏度下用温和的旋转孵育利沙酸盐一小时。在此稀释20卷稀释缓冲液中的酸盐，并在15毫升的解流管中将其分成15毫升馏分，分子截止量为10千吨。使用摆动铲斗旋转器，在 4000 倍 G 和 4 摄氏度下旋转管约 15 分钟，直到每个分数集中在小于 1 毫升的体积。在每个浓缩的利沙酸盐馏分中加入14毫升稀释缓冲液，并重复浓缩过程。然后结合分数，使用前面描述的浓度过程将它们浓缩成六毫升的体积。首先准备文本协议中概述的反应组合。将此反应混合物添加到预清除的六毫升利沙酸盐中，并混合良好。加入162.5微升的还原试剂，混合好。在室温下以800转/时在轨道摇床上孵育两小时。在此淬火后，加入5毫摩尔EDTA，并在室温下在轨道摇床上孵育5分钟。接下来，将反应混合物分成两个50毫升的管子，每根管中含有四卷预冷却的甲醇，并在负30摄氏度下孵育30分钟。在4000倍G和4摄氏度的离心机15分钟。丢弃上流液，在颗粒中加入一至两毫升预冷却甲醇。上下移液以分解颗粒，确保颗粒最终完全悬浮，没有可见的块。重复离心和再暂停过程两次。在此之后，仔细抽出残留的甲醇，确保不干扰颗粒。加入10毫升的重组缓冲液，上下移液溶解颗粒。在4000倍G和4摄氏度的离心机10分钟。然后将上一液转移到新管中，确保收集 20 微升样品进行质量控制。将已清理和重组的样品转移到准备好的链球菌珠上。在四摄氏度下孵育，温和旋转过夜。在 4000 次 G 下旋转珠子五分钟。将上一液转移到新管中，确保收集 20 微升样品进行质量控制。用 10 毫升洗涤缓冲液 A. 在转速 12 rpm 和室温下温和旋转 10 分钟，清洗珠子。然后在4000次G下旋转珠子5分钟。取出上一液，再次重复洗涤和离心过程。重复此洗涤过程以洗涤缓冲液 B 和洗涤缓冲液 C，如文本协议中所述。在此之后，用10毫升50毫升盐酸三酯洗涤珠子。将珠子在 4000 次 G 下旋转五分钟。拆下上流液，将珠子均匀地分割在两个 1.5 微离心管之间。要开始躲避捕获的 RNP，在 400 微升洗涤、定居的珠子中加入 400 微升准备好的生物素洗脱缓冲液。在室温下以1500转/分的速度在轨道摇床上孵育20分钟。然后在轨道摇床上孵育，热块在1500 rpm和65摄氏度，持续10分钟。将珠子在 7800 倍 G 下离心一分钟，然后收集 elud 的 RNP。向珠子添加400微升新鲜生物素洗脱缓冲液。重复孵化和离心过程，收集第二个耳，并将两个耳管组合在一个15毫升管中。接下来，在组合的 RNP 流液中添加三卷稀释缓冲液，以降低 SDS 的浓度。使用 0.5 毫升超滤管，将样品浓缩到文本协议中概述的约 40 微升。每微升 RNase A 添加 0.5 微克，并在 37 摄氏度下孵育两小时，以释放交对的 RNase 中的 RBP。收集两个微升的 RBC 进行质量控制。在优化 CARIC 协议时，质量控制步骤至关重要。通过凝胶荧光分析对点击标记样品进行具有代表性的质量控制。只有双标记的样本在高分子量下显示一个强烈的涂片带，表示交联 RNP 的信号。通过省略四个 SU 或 EU 来取消 RNP 信号。它也可以通过消化与RNase A.In在某些情况下，在没有四个SU控制样本中观察到一个强大的涂抹带。此频段表示标记的未交叉链接 RNase，在大多数情况下，该 RNase 在热加热期间会降解。通过西方印迹分析评估亲和力民意测验数据缺陷的质量控制，分析在下拉和下拉后显示样品中的生物素信号。对捕获的RSP进行银染色分析后发现，对于 HeLa 细胞，一般总捕获效率在输入蛋白的 0.05% 到 0.1% 之间。在尝试此过程时，重要的是要记住，RNase 对核糖核素敏感。保持实验环境尽可能干净，以减少RNase的去生长。按照这个程序，使用质谱法进行蛋白质阿拉木图鉴定，可以预造铁结合蛋白。开发后，CARIC 技术将为更全面地了解工作中邮政转录 OT 法规铺平道路。

Research

JoVE Journal

Biochemistry

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

mRNA Interactome Capture from Plant Protoplasts

mRNA从植物原生质体捕获

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

使用阿基-树脂辅助捕获检测蛋白质S-囊肿

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support