我们非常感谢技术支持四逆Miettinen，明娜Pöllänen和塔如Rautavesi的。我们感谢赫尔卡Nurkkala提供的HEK 293细胞中表达的双链球菌标记的GFP和佩卡Evijärvi提供录音设备。这项工作是由芬兰科学院，授权号码138329，134684和258978。

1，交联链球菌-Tactin聚甲基丙烯酸酯树脂与双（磺基琥珀酰亚胺）的辛二酸（BS3） <ol><li>平衡含有2mg BS3交联剂的室温1密封微管中。注意：BS3是一种有害物质。戴防护手套和护目镜。 </li><li>重悬链球菌-Tactin聚甲基丙烯酸酯树脂（在100毫米的Tris-HCl，pH 8.0的50％悬浮液，1 mM的EDTA，150 mM氯化钠）进行了简短的剧烈震荡，并立即用枪头与600微升悬浮液转移到离心柱结束切断。 </li><li>离心机在1500×g离心30秒，在室温下进行。弃去流通，并添加450微升磷酸盐缓冲盐水（PBS）中的列。 </li><li>重复前面的步骤2次以上，以完全代替Tris缓冲液，用PBS和离开该树脂在430微升PBS的最后离心后调节pH值至8.0。 </li><li>穿刺微管的铝箔与BS3与含有100的移液管尖1;升超纯水。通过轻轻地上下吹打溶解BS3粉末在水中，并立即加入20微升的溶液中，以旋转柱。 BS3在交联反应中的终浓度为〜1.2毫米。 </li><li>旋转30分钟，在室温下的列。检查该树脂与BS3溶液适当混合。 </li><li>以淬灭反应，加入6微升3M的Tris-HCl，pH值7.5和旋转另一个15分钟，在室温下的列。 </li><li>离心机在1500×g离心30秒，在室温下进行。弃去流通和重悬的交联树脂在450微升的Tris缓冲盐水与吐温20（TBST）。重复离心和洗涤步骤两次以上。在最后一步中，重悬树脂在450微升TBS的。 </li><li>用枪头末端的树脂悬液转移至新管切断。重悬在左与另一个450微升TBS的列中的树脂和传送到同一管中。重复次e最近步骤一次以上，以确保该树脂从柱的最大传送到管内。 </li><li>让管静置10分钟，在室温下和通过除去过量的TBS将音量调节到600微升。该树脂可立即使用（推荐），也可以保存在4°C，无冻结。 </li></ol>在双链球菌标签的诱饵蛋白及相关配合物的2。绑定到交联链球菌-Tactin聚甲基丙烯酸酯树脂<ol><li>离心机1毫升在结合缓冲液以17,000×g离心10分钟，在4℃下，将上清液转移到新管中的诱饵蛋白的样品。 </li><li>为了最大限度地减少内源性生物素化的蛋白的链球菌-Tactin树脂的结合中，添加抗生物素蛋白为100μg/ ml的终浓度，并在4℃下旋转15分钟<ol><li>如果从步骤1.10的交联树脂已被存储为在延长的时间期间内，在4℃，离心，在400×g离心收集FO树脂在4℃下，R 1分钟，弃去上清液并用1ml的TBST洗涤树脂。重复离心和洗涤步骤2次以上，第一次用TBST，然后用TBS。离心管中，在400×g离心1分钟，在4℃，以除去过量的TBS调整到原来的体积。 </li></ol></li><li>通过涡旋重新悬浮交联链球菌-Tactin树脂。立即加入50微升树脂悬浮液的使用切割枪头含有诱饵蛋白质样品的管中，在4℃下旋转，另外的30分钟</li><li>在等待时，恒温设定为55℃，预热500微升的洗脱缓冲液对在步骤3.1中使用。 </li><li>离心机在400×g离心1分钟，在4℃下弃去上清液，并在4℃下用1ml预冷的洗涤缓冲液＃1的洗涤树脂在旋转器上5分钟。重复离心和洗涤步骤3次。在最后一步中，重悬在树脂中加入250μl的洗涤缓冲液＃2。 </li><li>风帆<html> ansfer树脂悬浮于新鲜的自旋柱。重悬残留在管中的另一个250微升的洗涤缓冲液＃2的树脂和传送到同一列中。 </li><li>离心机在400×g离心3分钟，在4℃，弃去流通和柱转移到新鲜海豚机头2毫升管。立即进行下面的洗脱步骤。 </li></ol>具体蛋白复合物3。洗脱<ol><li>从步骤2.4加入150μl预热的洗脱缓冲液中，以旋转柱。 </li><li>在孵育恒温5分钟，55℃，摇在1400转每分钟。 </li><li>离心机在1500×g下在室温下放置1分钟。 </li><li>丢弃的列和存储纯化的目的蛋白在≤-20℃。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="598"><tbody><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;">磷酸盐缓冲盐水（PBS）中</td></tr>0“&gt; <td height="20" style="height:20px;width:299px;">的Na 2 HPO 4 </td><td style="width:299px;"> 10毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> KH 2 PO 4 </td><td style="width:299px;"> 2mM的</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">氯化钠</td><td style="width:299px;"> 137毫</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">氯化钾</td><td style="width:299px;"> 2.7毫米</td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;">将pH调节至7.4，除非另有说明（pH值8.0 1.4节） </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tris缓冲盐水（TBS） </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">的Tris-HCl，pH 7.4的</td><td style="width:299px;"> 50毫米</td></tr><tr height="20"><td hei<html> GHT =“20”的风格=“高度：20像素，宽度：299px;”&gt;氯化钠</td><td style="width:299px;"> 150毫</td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;">三缓冲盐水与吐温20（TBST）的</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">的Tris-HCl，pH 7.4的</td><td style="width:299px;"> 50毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">氯化钠</td><td style="width:299px;"> 150毫</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">吐温20 </td><td style="width:299px;"> 0.1％（体积/体积） </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;">结合缓冲液</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">的Tris-HCl，pH 8.0的</td><td style="width:299px;"> 25毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">氯化钠</td>X上;“&gt; 550毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">氟化钠</td><td style="width:299px;"> 5毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0.5毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">甘油</td><td style="width:299px;"> 10％（V / V） </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0.1毫米</td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;">洗涤缓冲液＃1 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">的Tris-HCl，pH 8.0的</td><td style="width:299px;"> 25毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">氯化钠</td><td style="width:299px;"> 500毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">氟化钠</<html> TD&gt; <td style="width:299px;"> 5毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0.4毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> IGEPAL CA-630 </td><td style="width:299px;"> 0.2％（体积/体积） </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">甘油</td><td style="width:299px;"> 5％（体积/体积） </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0.1毫米</td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;">洗涤缓冲液＃2 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">的Tris-HCl，pH 8.0的</td><td style="width:299px;"> 25毫米</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">氯化钠</td><td style="width:299px;"> 150毫</td></tr><tr height="20">2“高度=”20“的风格=”高度：20像素，宽度：597px;“&gt;洗脱缓冲液</td></tr><tr height="20"><td height="20" style="height:20px;width:299px;">的Tris-HCl，pH 8.0的</td><td style="width:299px;"> 25毫米</td></tr><tr height="22"><td height="22" style="height:22px;width:299px;"> SDS </td><td style="width:299px;"> 1％（重量/体积） </td></tr></tbody></table> 表1在本研究中使用的缓冲器。

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没有利益冲突的声明。

上述协议可用于纯化目的和其相关的复合物中的任何合适的缓冲液不含有生物素或强变性剂的任何双链球菌标签的诱饵蛋白。在该协议的当前版本中，结合和洗涤都相对严格的条件下在高盐和非离子型去污剂的存在下进行。尽管这会导致更少的背景，脆弱的蛋白质复合物可以解离这些条件下。为了保持这种低亲和力复合物，盐的浓度可被降低和非离子型洗涤剂，可减少或省略的结合和洗涤缓冲液?...

近年来，Strep-标签技术已成为广泛使用的生物医学研究中的许多领域，包括蛋白质组学和结构生物学。这种蛋白质纯化技术，这依赖于重组蛋白的融合到一个短的Strep-标签肽，已经成熟账面链球菌-Tactin，链霉的具有改进的肽结合的能力的基因工程变体亲和基质的到来。1，2含有两个拷贝Strep-标签II的指定双Strep-标签或SIII-标签，表现出对链球菌-Tactin基质比那些仅含有一个单一的Strep-标签，确保了重组蛋白质的更有效的纯化具有更高的亲和力和融合蛋白其相关的具有约束力的合作伙伴。然而，双链球菌标签蛋白对链球菌-Tactin较高的亲和力也有其不利的一面。这种蛋白质与过量的生物素的竞争性洗脱可能不完整，导致减少靶蛋白的产量。 A M矿石有效的替代方案是洗脱SDS，但它会导致与链球菌-Tactin不需要的样品污染从树脂上释放，使得用蛋白质组学分析测定不相容。本文提出了一种技术，通过第一稳定链球菌-Tactin的通过化学交联的树脂偶联的四聚体，然后用SDS洗脱从所得的交联树脂的双链球菌标记的蛋白质和其相关的复合物来克服这种限制。因此，足够的蛋白质产量可在不与链球菌-Tactin样品污染来实现，从而允许通过质谱法进一步分析。 该方法适用于任何重组融合蛋白的纯化与表面暴露SIII-标签3或双-Strep-标签（氨基酸序列WSHPQFEK（GGGS）3 WSHPQFEK和SAWSHPQFEK（GGGS）2 GGSAWSHPQFEK，分别）。该蛋白质可以是动物，植物或细菌来源的，可以从任一总细胞中分离裂解物或富集的细胞器部分。作为一个例子，我们在这里描述一个SIII标签蛋白VPG-Pro的马铃薯A病毒（PVA）4从PVA-感染的烟草本塞姆氏烟草植物的细胞核部分的纯化。如前所述5，具有以下修饰的核成分进 ​​行分离：细胞没有用甲醛处理，丁酸钠代替在所有的缓冲区用5mM氟化钠，完全蛋白酶抑制剂被置换为PMSF，加入Triton X-100的浓度在萃取缓冲器＃2降低到0.3％（体积/体积），并通过蔗糖垫离心得到（提取缓冲液＃3）的核沉淀再悬浮于1.45毫升预冷的结合缓冲液中并旋转1.5小时，在4℃下根据下面描述（见第2部分）的协议包含SIII标签的诱饵蛋白和相关的复合物（诱饵蛋白样品）所得的核提取物进行了处理。

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr>
			<td>Bis(sulfosuccinimidyl) suberate (BS3), No-Weigh format, 8 x 2mg</td>
			<td>Pierce/Thermo Scientific</td>
			<td>21585</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com CAUTION: Hazardous substance. Causes serious respiratory, skin and eye irritation. Wear protective gloves and eye protection.</a></td>
		</tr>
		<tr>
			<td>Strep-Tactin MacroPrep resin (50% suspension)</td>
			<td>IBA</td>
			<td>2-1505</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter, cellulose acetate membrane, pore size 0.45 &#956;m, non-sterile</td>
			<td>Costar (Corning)</td>
			<td>8163</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Dolphin-nose tubes</td>
			<td>Costar (Corning)</td>
			<td>3213</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Avidin&#160;</td>
			<td>IBA</td>
			<td>2-0204</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
	</tbody>
</table>

one-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (bs3)

链球菌标记的融合蛋白对携带一个精心设计的链亲和素（链霉素-Tactin）树脂亲和纯化已成为生理条件下广泛使用的方法为蛋白质复合物的分离。含有两个拷贝Strep-标签II的指定双Strep-标签或SIII-标签，融合蛋白具有对链球菌-Tactin相比，那些仅含有一个单一的Strep-标签更高亲和力的优点，因而允许更有效的纯化。然而，这一优点是通过一个事实，即双链球菌标记的蛋白与生物素的洗脱可能不完整，导致低蛋白恢复抵消。回收可以通过使用变性洗脱剂十二烷基硫酸钠（SDS）得到显着改善，但是这会导致样品污染链球菌-Tactin从树脂上释放，使得与下游蛋白质组分析测定不相容。为了克服这种限制，我们已经开发出一种方法，其中树脂耦合链球菌-Tactin的四聚体首先，通过共价交联与双（硫代琥珀酰亚胺基）辛二酸酯（BS3），将所得的交联树脂，然后用纯化靶蛋白复合物在单一批次纯化步骤稳定化。高效洗脱SDS确保了良好的蛋白质回收，而没有污染链球菌-Tactin允许通过质谱下游蛋白分析。作为概念验证，我们在这里描述的协议为SIII标记病毒蛋白VPG-PRO从病毒感染N的核提纯使用链球菌-Tactin聚甲基丙烯酸酯树脂的交联与BS3 本塞姆氏烟草植物。相同的协议，可用于净化任何感兴趣的双链球菌标记的蛋白质和其生理结合配偶体进行表征。

纯化过程在图1中示意性地示出，以与其他现有的纯化方法相关联的问题的表示一起。 <img alt="图1" fo:content-width="5.5in" src="/files/ftp_upload/51536/51536fig1highres.jpg" width="650" /> 图1的纯化过程的示意图。的工作流程包括通过共价交联与BS3，双-链霉素标记的诱饵蛋白和相关的复合物结合到交联的树脂的树脂偶联链球菌-Tactin四聚?...

Watch this Scientific Journal Video about One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3 at JoVE.com

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

的方法被描述为在链亲和素修饰的双链球菌标记的融合蛋白和它们的特定配合物的净化效率（链球菌-Tactin）树脂共价交联的双（硫代琥珀酰亚胺基）辛二酸酯（BS3）。该方法具有速度快，良好的靶蛋白回收率和纯度高的优点，并且与随后的分析通过质谱兼容。

对链球菌-Tactin树脂交联的双（磺基）辛二酸酯（BS3）双链球菌标记蛋白及其复合物的一步纯化

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

one-step-purification-twin-strep-tagged-proteins-their-complexes-on

Research

JoVE Journal

Biology

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

18.6K 次观看.  University of Helsinki. 的方法被描述为在链亲和素修饰的双链球菌标记的融合蛋白和它们的特定配合物的净化效率（链球菌-Tactin）树脂共价交联的双（硫代琥珀酰亚胺基）辛二酸酯（BS3）。该方法具有速度快，良好的靶蛋白回收率和纯度高的优点，并且与随后的分析通过质谱兼容。 

视频: One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

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 ...

科研

生物学

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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 ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

一步代谢：碳水化合物，在一个单一的过程量化的有机和氨基酸

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

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 ...

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 ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

冷冻干燥细菌及其影响细胞生存的配方

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...