这项工作是由加拿大创新基金会（CFI），加拿大卫生研究所（CIHR）学院，以及自然科学与工程研究理事会（NSERC），加拿大的赠款支持。

第1部分：中断一个流感病毒粒子<ol><li>添加到一个贝克曼聚碳酸酯离心管，设计成TLA - 120.2转子适合在贝克曼的Optima使用（11毫米× 34毫米）的750名产妇和新生儿破伤风缓冲液（MES的20毫​​米，150毫米氯化钠，30毫米的Tris，pH值7.5）马克斯 - E超速离心机。 </li><li>添加到该管的A型流感病毒（H3N2型X - 31 A/AICHI/68应变; 2毫克/毫升）500μL。移液器向上和向下几次与产妇和新生儿破伤风的缓冲区混合病毒。 </li><li> 109000 XG，4 ° C离心10分钟，在一个贝克曼舰最大- E使用TLA - 120.2转子离心机。 </li><li>去除上清，重新悬浮颗粒在500μL中断缓冲区（100毫米氯化钾，氯化镁2 5毫米，5％（W / V）甘油，50毫米octylglucoside，10毫克/毫升溶血卵磷脂，二硫苏糖醇1.5毫米，100毫米的MES， pH值5.5）。 </li><li>涡大力，然后摇在31℃20分钟在一个小指thermomixer彗星。 </li></ol>第2部分：甘油梯度泥沙速度离心<ol><li>准备放入一个贝克曼ultraclear离心管（13毫米× 51毫米）以下的甘油，甘油梯度：1毫升70％（V / V）甘油，0.75毫升50％甘油，0.375毫升40％甘油，和1.8毫升33％甘油。甘油的解决方案是由纯甘油的混合纳米缓冲区（MES，pH值5.5，50毫米150毫米氯化钠）。还准备同时含有甘油和缓冲的平衡管。 </li><li>旋涡再次扰乱了病毒样本，并加载到甘油梯度。 </li><li>离心机梯度为3.75小时，217000 XG 4 ° C，在一个贝克曼MLS - 50斗摆动转子。 </li><li>离心完成后，手动收集250μL等份，从管的顶部开始梯度。保持在冰上或在4 ° C的分数</li></ol>第3部分：通过SDS聚丙烯酰胺凝胶电泳分析的甘油梯度分数<ol><li>从每个几分新鲜管取出20μL。 </li><li>新增5 5倍的SDS - PAGE样品缓冲液。 </li><li>加热至95 ° C 5分钟。 </li><li>简要离心样品，并加载到10％的聚丙烯酰胺凝胶，分子量标记，其中包括在井之一。 </li><li>凝胶上运行的样本，直到溴酚蓝上样染料达到接近凝胶底部。 </li><li>考马斯蓝染色的凝胶。 </li></ol>第4部分：vRNP分数的浓度<ol><li>选择含有主要NP甘油分数，将它们结合起来，和他们分配到两个贝克曼聚碳酸酯离心管（11毫米× 34毫米）。 </li><li>焦碳酸二乙酯（DEPC）处理过的超纯水，和吸管起来，几次下来混合填充每个管。 </li><li> 157000 XG，4 ° C离心4.5小时，在一个贝克曼TLA - 120.2转子。 </li><li>去除上清液，并在50μLDEPC处理过的水重新悬浮沉淀。 </li><li>如果需要，集中vRNPs 280可以衡量的。为NP是汇集分数的重要组成部分目前，估计的NP摩尔可以使用 M -1 厘米 -1（通过ProtParam 3确定）的55350消光系数计算。 </li><li> vRNPs纯化，分装和存储他们冻结在-80 ° C为以后使用。 </li></ol>第5部分：vRNPs负染<ol><li>到9μL稀释1μL纯化vRNP新鲜和过滤两次产妇和新生儿破伤风的缓冲区（三30毫米，20毫米的MES，150 mM氯化钠，pH值7.5）。其他缓冲区也确定淡化vRNPs洗试样的网格（步骤5.5），但如果不使用PBS醋酸铀负染。 </li><li>辉光放电的标本（铜透射电镜）电网之前30秒一个parlodion和碳薄膜的涂层。 </li><li>保持与镊子新鲜标本电网辉光放电，并应用到标本电网5μL稀释vRNP下降。 </li><li>保留标本8分钟电网下降。 </li><li>在等待，在板凳上的封口膜小片地方，并免除2滴10μL每个产妇和新生儿破伤风缓冲区（洗电网），和一个新鲜的染色溶液80μL下降（1％钼酸铵或1 ％醋酸双氧铀）上的封口膜。 </li><li>吸附的8分钟后，灯芯从样品溶液的滤纸标本网格（切成三角形）的一部分。不要让干的标本电网。 </li><li> 2含1分钟的总时间为产妇和新生儿破伤风缓冲区各10μL滴标本电网清洗。这样做是通过仔细的下降降低试样的网格，然后排汗关闭部分样品溶液的滤纸标本网格（而不让试样电网干）。 </li><li>后立即排汗的缓冲区从标本电网的最后一滴，完成LY淹没的标本染色液滴内的电网，并等待1分钟。 </li><li>完全灯芯染色滤纸标本电网解决方案。 </li><li>允许风干的标本透射电子显微镜下观察前几分钟电网。 </li></ol>第6部分：代表性的成果： <img src="/files/ftp_upload/old/1105_2009_1_22_1/1105_Figure2.png" alt="图2" width="600" height="215" />图1 由于A型流感NP（〜56 kDa）的蛋白在病毒核蛋白复合物中发现的主要，NP带一般是最强的乐队在vRNPs（图1）的分数。除了到NP，每个流感vRNP还包含一个三聚体的RNA聚合酶（82，86和86.5 kDa的分子量）复杂的副本。这些可能会或可能不会考马斯亮蓝染色可见的，因为他们的丰度相比是低的NP。然而，聚合酶，可以检测到，而不是一个Commassie蓝染色，凝胶进行银染。流感基质蛋白M1（〜28 kDa）的，应最低限度在目前存在的NP蛋白峰分数的分数。 <img src="/files/ftp_upload/old/1105_2009_1_22_2/1105_3.png" alt="图3" width="600" height="360" />图2 负染色透射电子显微镜下观察vRNPs应该产生类似于可变长度的杆状颗粒约30纳米，长度为120纳米（图2a）vRNPs。低聚NP是一个NP的分子链进一步折叠成一个双螺旋重复结构的组织，如此循环，有时可以看到（图2b），这些杆状颗粒的两端。

<ol>
	<li>Kemler, I., Whittaker, G., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+microinjected+influenza+virus+ribonucleoproteins.">Nuclear import of microinjected influenza virus ribonucleoproteins.</a> Virology. 202, 1028-1033 (1994).</li><li>Wu, W. W. H., Weaver, L. L., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+the+nuclear+localization+sequences+on+influenza+a+ribonucleoprotein+complexes.">Ultrastructural analysis of the nuclear localization sequences on influenza a ribonucleoprotein complexes.</a> J Mol Biol. 374, 910-916 (2007).</li><li>Gasteiger, E., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Wu, W. W. H., Sun, Y. H. B., Pant&#233;, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+influenza+A+viral+ribonucleoprotein+complexes+is+mediated+by+two+nuclear+localization+sequences+on+viral+nucleoprotein.">Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein.</a> Virol J. 4, 49-49 (2007).</li><li>Babcock, H. P., Chen, C., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single-particle+tracking+to+study+nuclear+trafficking+of+viral+genes.">Using single-particle tracking to study nuclear trafficking of viral genes.</a> Biophys J. 87, 2749-2758 (2004).</li></ol>

vRNPs净化Kemler 等人 （1994年）所描述的程序的基础上，我们和其他人也用此协议隔离vRNPs，研究它们的核进口。2,4,5 我们建议使用无RNase的技巧和操纵vRNPs管时，因为病毒基因组RNA的组成，因此很容易降解的RNA的存在。此外，所有的缓冲区，应即核糖核酸免费的水。这里所描述的协议，是一种酸性提取（如中断缓冲区和甘油梯度的pH值是5.5）。同样，一个基本的?...

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>H3N2 X-31 A/AICHI/68 influenza A</td>
<td>&nbsp;</td>
<td>Charles River Laboratories</td>
<td>490715</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M-8250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G33-1</td>
<td>&nbsp;</td>
<tr>
<td>Octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O-8001</td>
<td>&nbsp;</td>
<tr>
<td>Lysolecithin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-4129</td>
<td>&nbsp;</td>
<tr>
<td>Dithiothreitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-9779</td>
<td>&nbsp;</td>
<tr>
<td>Coomassie brilliant blue G-250</td>
<td>&nbsp;</td>
<td>Kodak</td>
<td>1367796</td>
<td>&nbsp;</td>
<tr>
<td>diethyl pyrocarbonate (DEPC)-treated water</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>750024</td>
<td>&nbsp;</td>
<tr>
<td>Uranyl Acetate</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>19481</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Molybdate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A-674</td>
<td>&nbsp;</td>
<tr>
<td>Optima MAX-E Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>434491</td>
<td>&nbsp;</td>
<tr>
<td>MLS-50 Rotor Package, Swinging Bucket</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>367280</td>
<td>&nbsp;</td>
<tr>
<td>TLA-120.2 Rotor Assembly, Fixed-Angle, Titanium</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362046</td>
<td>&nbsp;</td>
<tr>
<td>Eppendorf Thermomixer</td>
<td>&nbsp;</td>
<td>Brinkman</td>
<td>022670000</td>
<td>&nbsp;</td>		</tbody>
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purification and visualization of influenza a viral ribonucleoprotein complexes

A型流感病毒的基因组由8负意义上说，单链RNA分子单独包装，与A型流感到的病毒ribonulceoprotein颗粒（vRNPs）核蛋白（NP）的多个副本。流感vRNPs封闭的病毒包膜内。然而，在进入细胞，这些vRNP复合物释放到细胞质，在那里他们获得主机核运输机械。为了研究核进口的流感vRNPs和流感基因组复制的，它与孤立的vRNPs工作，使病毒的其他组件不干扰这些进程是有益的。在这里，我们描述一个过程来净化这些vRNPs从A型流感病毒。程序启动以释放笼罩病毒粒子vRNP复合物，用清洁剂A型流感病毒粒子的干扰。 vRNPs是从A型流感病毒粒子在33-70％的速度沉淀间断甘油梯度的其他部分隔开。从甘油梯度获得的分数，然后通过SDS - PAGE电泳分析，考马斯亮蓝染色后。包含NP峰值分数，然后汇集在一起​​，并通过离心浓缩。浓度后，vRNPs的完整性是通过可视化的传输电子显微镜负染色后的vRNPs验证。甘油梯度纯化的修改，从Kemler<em&gt;等。</em&gt;（1994）<sup&gt; 1</sup&gt;和负染色进行吴<em&gt;等。</em&gt;（2007年）。<sup&gt; 2</sup

Watch this Scientific Journal Video about Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes at JoVE.com

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

A型流感病毒的基因组由8个独立的复合物，RNA和蛋白质，称为病毒核蛋白复合物（vRNPs）。本文介绍的甘油梯度纯化和透射电子显微镜A型流感vRNPs可视化。

A型流感病毒的核蛋白复合物的分离纯化及可视化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

purification-visualization-influenza-viral-ribonucleoprotein

Research

JoVE Journal

Biology

12.9K 次观看.  University of British Columbia - UBC. A型流感病毒的基因组由8个独立的复合物，RNA和蛋白质，称为病毒核蛋白复合物（vRNPs）。本文介绍的甘油梯度纯化和透射电子显微镜A型流感vRNPs可视化。

视频: Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

制备，纯化和磁共振成像造影剂的使用稀土配合物的表征

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

产生和人类INO80染色质重塑复合物及其亚纯化

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...