Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH under award number R01AI114362 and Welch Foundation grant I-1782 to Iv&#225;n D'Orso.

1.电镀细胞<ol><li>转染前一天，板3.5×10 6个 HEK293T细胞于10ml Dulbecco改良的Eagle培养基（DMEM）中的补充有10％（体积/体积）胎牛血清（FBS）和1％（体积/体积）青霉素/链霉素（10,000 U / ml的股票）每100毫米的菜。板3 - 5 100个实验/条件毫米的菜肴，以保证足够的蛋白质回收。 注:板的数量为每个实验/条件，这将依赖于感兴趣的蛋白质复合物的表达水平和溶解性来确定。可替代地，如果需要更大规模的纯化，细胞系可以适于在悬浮于生长在旋转烧瓶2中 ，但这种方法不会对瞬时转染工作。 </li></ol> 2.转染细胞或诱使稳定细胞系<ol><li>第二天，检查在显微镜下细胞。细胞必须是70 - PROCE前80％汇合eding。 </li><li>转染的细胞以每100mm培养皿转染试剂的质粒DNA的7微克和21微升总混合物。转染细胞的载体表达GFP（ 表1）作为转染效率的对照。 注意:如果使用诱导响应于多西环素14，15的两个或更多个复杂亚基表达的HEK293-T-REX或类似的稳定细胞系，该诱导剂将必须加入到细胞中，并培养48小时。同样地，也将需要验证目的诱导的GFP在加入诱导剂的表达的稳定细胞系。继续执行步骤2.7。 </li><li>准备每100mm培养皿转染混合物。在1.5ml微量离心管中，总质粒DNA（对应于两个或更多的质粒）的7微克混合500μl的未补充的DMEM。在一个单独的离心管中，混合500微升未补充的DMEM的Wi日21微升的转染试剂。重复每个转反应。 </li><li>吸取转染试剂溶液到质粒DNA溶液（不混合以相反的顺序解）。吹打至少4次混合。 </li><li>孵育该混合物在室温下进行10 - 15分钟，但超过15分钟不再。 </li><li>倾斜板创建介质贮存器和小心逐滴吸取转染混合物到板的内部侧而不会干扰细胞。返回菜到原来的平面位置和漩涡轻轻分配混合物。 </li><li>孵育在37℃下将细胞在5％的CO 2的潮湿的细胞培养箱中（该条件在下文中称为"组织培养孵化"）。更换介质5小时转染后（可选）。 </li></ol> 3.检查转染或诱导效率<ol><li>在24小时后转染，可视化流感下细胞orescent显微镜检查转染效率（或表达GFP控制HEK293T-REX稳定细胞系的诱导）。孵育另外24小时。 </li></ol> 4. STREP亲和纯化（STREP AP） <ol><li> 收集的细胞 <ol><li>在48小时后转染，删除媒体并加入8毫升冷1X磷酸盐缓冲液（PBS）的。吸管上下从板脱落的细胞。 </li><li>收集和细胞悬液转移到15毫升管中，在4℃旋转细胞向下以800 xg离心4分钟。 </li><li>删除PBS上清。重复降速（800×g离心在4℃下1分钟），以消除任何残留的PBS中。保存细胞沉淀在-80℃以备将来使用或按照以下步骤继续蛋白纯化。 </li></ol></li><li> 裂解细胞 <ol><li>悬浮用5×细胞沉淀体积被动裂解缓冲液的细胞沉淀（每个板250微升）（PLB:20mM的Tris-盐酸pH为7.5，150 mM氯化钠，1.5mM的MgCl 2的，1毫摩尔DTT，蛋白酶抑制剂混合物片剂，5％体积/体积的甘油，和1.0％的NP-40）和细胞悬浮液转移到一个1.5 ml离心管。 </li><li>在4℃短暂涡旋悬挂和章动30分钟。 </li><li>降速细胞悬浮液以10,000×g离心10分钟，在4℃下。 </li><li>转移可溶性裂解物进入一个新的1.5 ml离心管并弃去细胞沉淀。可节省10％的可溶性裂解物作为最终体积的"STREP AP输入"。保存在4℃。 </li></ol></li><li> 平衡的STREP珠 注:使用STREP珠第一个AP一步！配合拉下来STREP珠复苏比那些FLAG珠拉下显著更多的蛋白质。 <ol><li>取出（正×40微升）+ n中的50％的STREP珠浆液微升（N =样品数量），并在4℃降速在1500 xg离心2分钟。 注:使用广泛米outhed提示，吸管珠。 </li><li>除去上清液，加入500μlPLB到珠。章动珠子混合物在4℃5分钟。降速在1500 XG为2分钟，4℃。 <ol><li>重复总共3次洗涤。重悬在PLB珠粒到n的终体积×40微升。 </li></ol></li><li>添加40微升50％的STREP珠浆液对每个细胞裂解物样品和章动2小时，在4℃。 </li></ol></li><li> STREP AP <ol><li>降速的解决方案在1500 XG为2分钟，在4℃。 </li><li>在4℃去除上清，店作为"STREP AP流通型（FT）"。 </li><li>加入500μlSTREP AP洗涤缓冲液I（20mM的Tris-盐酸pH值7.5，250mM的氯化钠，1.5mM的MgCl 2的，1毫摩尔DTT，5％甘油和0.2％的NP-40）的小珠的。章动珠子混合物在4℃，5分钟，离心以1500 xg离心在4℃下2分钟。弃去上清液。 <ol><li>重复，共4次洗涤。 <br/&gt;注:第四洗涤之前，将珠粒溶液转移至新的1.5 ml离心管，以减少蛋白质背景。这一步是必要的。 </li></ol></li><li>加入500μl洗涤缓冲液II（100mM的Tris-盐酸pH值8.0，150mM的NaCl和1mM EDTA）中并通过反相管平衡的珠子。降速在1500 xg离心2分钟，在4℃，弃上清。 </li><li>洗脱目的蛋白质用50μlSTREP洗脱缓冲液（100毫摩尔Tris-盐酸pH为8.0，150 mM氯化钠，1mM EDTA中，和2.5mM脱硫生物素）的。涡流在800rpm，在4℃下15分钟。 </li><li>降速珠粒在1500 xg离心1分钟，在4℃下。收集上清液。该级分对应于"STREP AP洗脱"样品。 </li><li>保存在4℃的珠粒级分用于第二洗脱，这可能会产生高达与第一洗脱获得的蛋白质的量的一半。在STREP AP洗脱分装10％的银染色和/或免疫印迹16。 </li></ol></li><li> 平衡的FLAG珠 <ol><li>取出（NX 16微升）+ N的FLAG珠溶液微升（N =样品数量），并在4℃降速在1500 xg离心2分钟。 注:使用广口提示，吸管珠。 </li><li>除去上清液，并添加FLAG洗涤缓冲液500微升（100毫摩尔Tris-盐酸pH为8.0，150 mM氯化钠，1mM的EDTA和0.1％NP-40）至珠。降速在1500 XG为2分钟，4℃。重复总共3次洗涤。 </li><li>重悬在FLAG洗涤缓冲液珠粒于nx 16微升的最终体积。 </li><li>在4℃加入16微升的FLAG珠浆剩余的STREP AP洗脱和章动过夜。 </li></ol></li></ol> 5. FLAG IP <ol><li>降速FLAG珠悬浮在1500 XG为2分钟，4℃。在4℃下保存上清液和存储为"FLAG IP流过"式（FT）。 </li><li>加50081; FLAG洗涤缓冲液将溶液的湖章动，在4℃下在4℃下5分钟，然后离心以1500 xg离心2分钟。重复，共4次洗涤和每次洗涤后除去上清液。 注:第四洗涤之前，蛋白质 - 小珠溶液转移至新的1.5 ml离心管，以减少背景。这一步是必要的。 </li><li>从最后的旋转除去上清液，并添加含FLAG肽的200毫微克/微升到珠粒FLAG洗涤缓冲液的30微升。涡流在800rpm，在4℃下2小时。 </li><li>降速悬浮液在1500×g离心2分钟，在4℃。收获在4℃将上清液和存储为"FLAG IP洗脱"。 注:洗脱样品可以通过银染色和/或免疫印迹使用标准协议16进行确认，并准备进一步蛋白分析。在建立免疫印迹，运行所有下面的示例:（1）STREP AP输入，（2）STREP AP FT，（3）STREP AP Eluti上，（4）FLAG IP FT，和（5）FLAG的IP洗脱。装卷将具有用于每个实验加以确定。所有收集的样品和珠子可以储存在4℃短期贮存，并在-80℃下长期贮存。 TAP样品可以通过质谱法进行分析，以核实它们的组件的身份，以确定新的翻译后后修饰，和/或到发现用纯化复杂2，17中的任何新的蛋白质相互作用。为了这个目的，在运行考马斯凝胶或银染色后，频带可使用干净的手术刀凝胶切。几个优秀条评论讨论质谱方法进行了先前公布的18，19。 </li></ol>

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The authors have nothing to disclose.

此处所描述的表达和从真核细胞中的蛋白质复合物的分离的协议不限于这样的分子组件的生化特性，但也可用于新的交互件和翻译后修饰，可以调节它们的功能的识别。亲和标签的使用并不限于是什么在这个协议中提到;但我们的经验表明，使用STREP作为第一个AP步骤显著增强了最终蛋白质 - 蛋白质复合物的产率。都与其它表位标签（ 例如 ，FLAG）相比，距离STREP珠的结合和洗脱步骤是非常有...

<html>蛋白质-蛋白质相互作用（质子泵抑制剂）是用于生物过程1，并在这些质子泵抑制剂进一步研究的精确调节临界可以告知他们的功能2。几种方法已被设计用于质子泵抑制剂的研究和表征以及用于新的调控蛋白组分的从头鉴定。 1989年，斯坦利领域和同事报道酵母双杂交（Y2H）检测3。这种方法允许在酿酒酵母作用因子（猎物）的定义的兴趣（诱饵）蛋白的公正和全面的鉴定。除了其用于发现质子泵抑制剂显着的效用，在Y2H测定可用于在酵母细胞中表征蛋白质对，限定最小相互作用域，以及识别废除这种相互作用的突变。通过修改Y2H测定中，质子泵抑制剂，也可在哺乳动物细胞中研究了类="外部参照"&gt; 4。在Y2H测定（ 如酵母三杂交系统）的变型也可以用于研究蛋白质的RNA，并在细胞中的蛋白质，小的有机配体的相互作用。 另一种常用的工具来研究质子泵抑制剂以同源系统是共免疫沉淀（共-IP）测定5。通过使用抗体免疫沉淀目的蛋白质，共同的IP测定使研究人员能够监测细胞对各种环境条件和实验条件质子泵抑制剂。使用表位标记的蛋白（ 例如 ，FLAG，Myc基因，STREP和HA，等等）在亲和纯化（AP）的方法，促进了从复合蛋白质混合物为几个下游测定法，包括免疫印迹蛋白质的分离，银染色的和酶分析。然而，这些以前的方法使能大量的蛋白质复合物的用于进一步表征，包括在体外在隔离ssays，通过质谱法，和翻译后修饰的识别调节亚基的发现。该AP方法的改进版本被称为串联的AP（TAP），这是通过创建与通过两个连续的AP 6,7纯化两个表位的融合蛋白研究质子泵抑制剂的纯化技术。在这篇文章中，我们提出了净化蛋白复合物，其中两个亚单位的标签与不同的表位，然后通过两个连续的AP（STREP AP随后FLAG IP）纯化TAP方法的变型。我们首先提供的TAP的一个简约概述（ 图1），然后所有的试验步骤的详细描述（ 图2），使得研究人员可以将它们应用到他们感兴趣的蛋白复合物。 为了证明在TAP方法的适用性，我们选择了一个良好表征细胞周期CDK络合物（简称PTEFB激酶），它是由所述调节亚基细胞周期蛋白T1（CycT1）和激酶（CDK9）的，并且是由RNA聚合酶II（RNA聚合酶II）8，9，10参与转录的调节。 P-TEFB磷酸聚合酶II和其相关联的消极延伸因子，这在启动子减轻转录暂停的C-末端结构域，并由此促进转录延伸11，12，13。考虑到这一众所周知的互动，STREP标记CycT1和FLAG标记CDK9均超过在HEK293T细胞中表达。互惠TAP实验与STREP标记CDK9和FLAG标记CycT1进行进一步验证该蛋白质相互作用是独立使用的表位。收集细胞并裂解48小时后转染。 （AP STREP之后FLAG的可溶性裂解液纯化TAPIP）。输入和纯化蛋白通过Western印迹和银染色（ 图3）分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Fisher Scientific</td>
			<td>SH30022FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30071</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Fisher Scientific</td>
			<td>MP091670049</td>
			<td></td>
		</tr>
		<tr>
			<td>PolyJet</td>
			<td>Fisher Scientific</td>
			<td>50-478-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail&#160;</td>
			<td>Roche&#160;</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr>
			<td>STREP-Tactin Superflow</td>
			<td>IBA</td>
			<td>2-1208-010</td>
			<td></td>
		</tr>
		<tr>
			<td>STREP-tag elution buffer</td>
			<td>IBA</td>
			<td>2-1000-025</td>
			<td></td>
		</tr>
		<tr>
			<td>EZview Red ANTI-FLAG M2 Affinity Gel</td>
			<td>Sigma</td>
			<td>SLBQ1981V</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 100mm &#215; 20 mm style Dish cell culture treated nonpyrogenic polystyrene 20/sleeve</td>
			<td>Corning&#160;</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Lo-Bind Eppendorf</td>
			<td>Fisher Scientific</td>
			<td>13-698-794</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Vortex Mixer</td>
			<td>Fisher Scientific&#160;</td>
			<td>02-215-370</td>
			<td></td>
		</tr>
		<tr>
			<td>48 hole micro tube foam rack</td>
			<td>Fisher Scientific</td>
			<td>02-215-386</td>
			<td></td>
		</tr>
		<tr>
			<td>Labquake shaker rotisserie&#160;</td>
			<td>Thermo&#160;</td>
			<td>415110</td>
			<td></td>
		</tr>
	</tbody>
</table>

tandem affinity purification of protein complexes from eukaryotic cells

活性的蛋白质-蛋白质和蛋白质-核酸复合物的纯化是用于酶活性和新颖性亚基和翻译后修饰的从头鉴定表征至关重要。细菌系统中允许的各种单个的多肽和蛋白复合物的表达和纯化。然而，该系统不使能包含翻译后修饰（ 例如 ，磷酸化和乙酰化）的蛋白质亚基的纯化，和新颖的调节亚基是只存在/在真核系统中表达的鉴定。在这里，我们提供一种新的，健壮的，并使用STREP-有效串联亲和纯化（TAP）方法和FLAG标签的蛋白质，有利于蛋白质复合物的纯化与来自真核细胞中瞬时或稳定表达表位标记的蛋白质的详细描述。此协议可应用到表征PROTEIN复杂的功能，以发现复杂亚基的翻译后修饰，并通过质谱分析法来识别新调节复杂的组件。值得注意的是，此TAP方法可用于研究真核或病原（病毒和细菌）组分形成蛋白复合物，从而产生一宽的下游实验机会阵列。我们建议，研究人员的蛋白质复合物的工作可以利用许多不同的方法这种方法。

在这篇文章中，我们展示了TAP方法充分表征CycT1-CDK9复合物（也被称为P-TEFB激酶）的适用性。 和CDK9-FLAG（CDK9:F），或CDK9-STREP（CDK9:S）和细胞周期蛋白T1-FLAG（CycT1:F）:编码细胞周期蛋白T1-STREP（S CycT1）质粒（ 表1），转染入HEK293T细胞。阴性对照包括用空载体和CDK9转染:F或CycT1:F质粒（ 图3A和B，</stron...

Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

tandem-affinity-purification-protein-complexes-from-eukaryotic

Research

JoVE Journal

Biochemistry

14.9K 次观看.  The University of Texas Southwestern Medical Center. We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

视频: Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

科研

生物化学

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

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 ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

利用 tap 标签纯化叶绿体转移蛋白配合物的亲和纯化

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

真核硼酸背液转运体的表达、增溶及纯化

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

亲和色谱法表达和纯化人基质金属蛋白酶-3

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.