提取移液器发展提供了资金部分由内部研究发展补助金，并授予的DRP。

1。样品裂解<ol><li>样品应在液态形式。如果样品是固体，例如食物或粪便样本，应暂停在液体介质，例如水或磷酸盐缓冲液（PBS）。 </li><li>混合样品500μL，500μL6M胍硫氰酸盐（pH值6.5）1.5 mL管。 </li><li>压低提取移液器灯泡，驱逐了周围的空气。 </li><li>拉入整个1毫升样品提取移液器，在吸附材料，通过让球慢慢地扩大。 </li><li>反转的提取工具等吸附剂珠和样品漏入灯泡。 </li><li>磨灯泡小心不要驱逐提取移液器样品样本的吸附剂珠。 </li></ol> 2。核酸提取（ 图1，A组） <ol><li>反转的提取移液器（开放向下）和驱逐到原来的1.5毫升管样品。 </li> &lt;李&gt;提取移液器拉进整个1毫升样品，通过在吸附剂，在一个适度的速度，小心地保持在颈部提取移液器吸附剂。 </li><li>按下灯泡驱逐到1.5 mL管整个样本。 </li><li>重复步骤2.2和2.3，通过吸附剂四次，共5科及格的样本。 </li><li>在吸附剂的第五通后，驱逐到的蛋白质提取液（15 mL锥形管）4ML整个1毫升样品，关闭管，留作以后使用。 </li><li> 1毫升95％的乙醇通过吸附剂的三倍以上，回国后，最后通过乙醇到其原始的管洗孤立的核酸。 </li><li>使用第二个1毫升，95％的乙醇洗，重复步骤2.6。 </li><li>按下和释放5分钟的灯泡，通过整个提取吸管周围空气干燥的核酸/吸附剂床。擦拭残留的乙醇提取移液器ü尖唱一个Kimwipe。 </li><li>通过250μL10毫米的Tris（pH 6.8）的吸附剂五倍恢复核酸。 Tris缓冲液，可以换成任何其他合适的，如PBS水溶液。最终的解决方案包含提取核酸。 </li></ol> 3。蛋白提取（图1，B组） <ol><li>混合反演2.5步的样品和蛋白提取液管，并通过在一个新的提取移液器吸附剂大约两到三年的这5毫升样品毫升。 </li><li>驱逐到同一个15mL的锥形管小心地保持在颈部提取移液器吸附剂整个样本。 </li><li>重复步骤3.1和3.2的样品，通过吸附剂的15倍以上。 </li><li>洗三次，喷射到它的原始管乙醇1毫升95％的乙醇通过吸附剂床的吸附剂和结合蛋白。 </li><li>使用第二个1毫升，95％的乙醇洗，重复步骤3.4。 </li><html><li>按下和释放5分钟的灯泡，通过整个提取吸管环境空气干燥蛋白/吸附剂床。擦拭残留的乙醇提取使用kimwipe吸管尖。 </li><li>恢复通过吸附剂五倍以上的PBS 250μL蛋白。 PBS缓冲液，可以换成任何其他合适的，如10毫米三（pH值6.8）的水溶液。最终的解决方案包含了样品的蛋白质含量。 </li></ol><img src="/files/ftp_upload/4202/4202fig1.jpg" alt="图1A" /> 图1。 <img src="/files/ftp_upload/4202/4202fig2.jpg" alt="图2" /> 图2。 <img src="/files/ftp_upload/4202/4202fig3.jpg" alt="图3" /> 图3。 <img src="/files/ftp_upload/4202/4202fig4.jpg" alt="图4" /> 图4。 内容“&gt; <img src="/files/ftp_upload/4202/4202fig5.jpg" alt="图5" /> 图5。

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	<li>Gupta, S. K., Keck, J., Ram, P. K., Crump, J. A., Miller, M. A., Mintz, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+data+gaps+pertaining+to+enterotoxigenic+Escherichia+coli+infections+in+low+and+medium+human+development+index+countries,+1984-2005.">Analysis of data gaps pertaining to enterotoxigenic Escherichia coli infections in low and medium human development index countries, 1984-2005.</a> Epidemiology and Infection. 136, 721-738 (2007).</li><li>Pawlowski, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+Pipette.">Extraction Pipette.</a> United States Patent and Trademark Office. , (2010).</li><li>Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheim-van Dillen, P. M., van der Noordaa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+simple+method+for+purification+of+nucleic+acids.">Rapid and simple method for purification of nucleic acids.</a> J. Clin. Microbiol. 28, 495-503 (1990).</li><li>Karalus, R. J., Pawlowski, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+and+Device+for+Isolating+a+Protein+Sample.">Method and Device for Isolating a Protein Sample.</a> United States Patent and Trademark Office. , (2010).</li><li>Huang, A., Friesen, J., Brunton, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+bacteriophage+that+carries+the+genes+for+production+of+Shiga-like+toxin+1+in+Escherichia+coli.">Characterization of a bacteriophage that carries the genes for production of Shiga-like toxin 1 in Escherichia coli.</a> Journal of Bacteriology. 169, 4308-4312 (1987).</li><li>O'Brien, A. D., Newland, J. W., Miller, S. F., Holmes, R. K., Smith, H. W., Formal, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shiga-like+toxin-converting+phages+from+Escherichia+coli+strains+that+cause+hemorrhagic+colitis+or+infantile+diarrhea.">Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea.</a> Science. 226, 694-696 (1984).</li><li>Wagner, P. L., Neely, M. N., Zhang, X., Acheson, D. W. K., Waldor, M. K., Friedman, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+a+Phage+Promoter+in+Shiga+Toxin+2+Expression+from+a+Pathogenic+Escherichia+coli+Strain.">Role for a Phage Promoter in Shiga Toxin 2 Expression from a Pathogenic Escherichia coli Strain.</a> Journal of Bacteriology. 183, 2081-2085 (2001).</li><li>Mizutani, S., Nakazono, N., Sugino, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+So-called+Chromosomal+Verotoxin+Genes+are+Actually+Carried+by+Defective+Prophages.">The So-called Chromosomal Verotoxin Genes are Actually Carried by Defective Prophages.</a> DNA Research. 6, 141-143 (1999).</li><li>Ptashne, 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> A Genetic Switch: Phage Lambda Revisited. , (2004).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2001).</li><li>Feng, P., Monday, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+PCR+for+detection+of+trait+and+virulence+factors+in+enterohemorrhagic+Escherichia+coli+serotypes.">Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes.</a> Mol. Cell. Probes. 14, 333-337 (2000).</li></ol>

没有利益冲突的声明。

在本报告所述的工具，化学和协议代表的电力，多场为基础的，点保健应用，可以利用的高分子萃取体系的第一个众所周知的例子。两种高分子类型，可以应用到下游的诊断或分析设备的数量。例如，孤立核酸PCR的质量（ 图2和3），而样品中的蛋白质成分是阳性（ 图4）。利用该萃取体系在严峻的世界或资源有限的地区，可以大大减少疾病的人口生活在那里的负担。此萃取体系也有助于在世界各地的疾病监测方案，通过提供一个强大而快速和成本有效的方法，在严峻或偏远地区的样品处理。 其中一个重要方面上文所述的提取方法是，它可以由用户修改。例如，在情况下，样本大小是大于或小于最初的500μLs（步骤1.2），用户可以调整相应的试剂量。也就是说，试剂的体积比为样本大小可以改变，而绝对量应保持不变。此外，在吸附剂的通道数的调整，也可以在用户的​​自由裁量权。例如，如果使用一个较大的样本量，通过吸附剂以上的上市时间（步骤2.4：3.3），样品将增加高分子型接触的吸附剂（核酸或蛋白质）的可能性。在通道的增加，应提高捕捉效率，直至达到吸附饱和。 我们发现，提取工具和相关的化学物质有一定的局限性，其中最重要的是恢复高度糖化蛋白萃取化学低效蛋白质。在事件下游的诊断针对的是一种糖蛋白，该系统可能无法识别的理想。此外，无论是核酸或蛋白质提取效率的上限和下限仍然被确定，正在进行和优化，以最大限度地回收效率。进一步发展的工具，将克服这些现有的知识差距。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>异硫氰酸胍</td><td> teknova </td><td> G4000 </td></tr><tr><td>乙醇</td><td> pharmco-Aaper </td><td> 11ACS200 </td></tr><tr><td> 2 - 丙醇</td><td> Sigma-Aldrich公司</td><td> 109827-1L </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A - 4503 </td></tr></tbody></table>

electricity-free, sequential nucleic acid and protein isolation

传统和新兴的病原体，如肠出血性大肠杆菌 （EHEC）， 鼠疫菌，或朊病毒疾病的重大关切，为政府，行业和世界各地的医疗专业人员。例如，相结合，与志贺氏菌 EHECs，每年约325,000名儿童的死亡负责，特别是在发展中世界的实验室为基础的识别，在美国常见的，是不可用1普遍。低成本的发展和分布，实地为基础，点保健工具，以帮助快速​​识别和/或病原体或疾病标志物的诊断可以大大改变疾病的进展和病人的预后。我们已经开发出一种工具来分离固相萃取（SPE）从样品中的核酸和蛋白质，没有电力或相关的实验室设备2。孤立的大分子，可用于diagno无论是在前进的实验室或使用现场设点的护理平台SIS。重要的是，这种方法提供了直接从联合国分裂的样本核酸和蛋白质数据的比较，通过对基因组和蛋白质组分析的佐证提供的信心。 我们的隔离工具，采用固相核酸分离的行业标准，繁荣技术，分离离液盐的解决方案，通常是异硫氰酸胍，通过结合基于硅粒子或过滤器3，核酸。 CUBRC专有的固相萃取化学，用于纯化蛋白，离液盐溶液在这种情况下，从废物或流通过继核酸隔离4。 由包装特色的化学成一个小，价格低廉，简单的平台，我们已经产生了核酸和蛋白提取的便携式系统，可以根据VAR进行iety的条件。孤立核酸是稳定的，可以运到一个位置，电源可用于PCR扩增，而蛋白质含量可以立即举行的手或其他免疫检测分析。在该领域的疾病标志物的快速鉴定，能显着改变指导适当的治疗过程中病情恶化的早期阶段，病人的结果。的工具和方法，适用于几乎任何传染性病原体使用，并为用户提供多高分子型分析的冗余，同时减少他们的后勤负担。

核酸分离：隔离核酸PCR扩增和蛋白污染的自由。 例1：回收的核酸适合PCR为基础的应用。例如， 图2显示了肠出血性大肠杆菌志贺毒素相关基因的扩增大肠杆菌 O157：H7大肠杆菌的EDL-933（ATCC＃35150）从核酸/蛋白的提取，使用提取移液器的过程。在这个和其他肠出血性大肠杆菌，志贺毒素STX 1 STX 2的基因被发现在温带的拉姆达样噬菌体，缺陷噬菌体控制下的P R的启动5 6 7 8 大肠杆菌菌株。在大肠杆菌中的SOS反应的激活大肠杆菌导致噬菌体的诱导和/或产生毒素9。我们利用丰等复用的PCR检测的修改后的版本1。1，以确定STX 1和STX 2基因在文化和尖刺的污水样本。车道和图2中的 B显示了未处理的文化样本（一个车道）的PCR扩增和处理文化样本（车道）的结果。这些结果是几乎相同的意义，有没有保真度损失在加工或孤立的核酸。接下来我们放大的尖刺未经处理的污水（双线四）或孤立核酸从尖刺污水酸（车道E和F）。这些数据表明，孤立核酸造成更大的与从尖刺污水样本的非隔离的核酸扩增。因此，我们得出这样的结论：要么PCR自然发现污水抑制剂被拆除后，隔离或核酸分离后集中。这两个假设的组合也是可行的。 未来紫外光谱表明，孤立核酸相对自由Øf污染蛋白。 图3显示了两个大肠杆菌的紫外光谱大肠杆菌 O157：H7型核酸酸提取，标记和隔离，隔离细菌培养的规范化谱，标记隔离前相比。孤立核酸的紫外吸收光谱类似于纯的核酸，有接近1.8 10 260 nm和280 nm之间的比例计算的吸收光谱。这些数据表明，有很少的污染蛋白质在核酸回收使用的技术描述部分。 蛋白质分离回收蛋白核酸/蛋白分离实验是纯蛋白质的免疫反应和电泳相同。 例2：分离蛋白组分应用的RapidChek E.大肠杆菌 O157横向流动检测条（SDIx上，公司）。控制显示在所有样品窝藏大肠杆菌阳性鉴定大肠杆菌<html> 0157，带或不带感应世腾（ 图4，分别车道乙和Ç）。 图4中的数据还显示，对于那些窝藏大肠杆菌样本的阳性结果大肠杆菌 O157核酸，然后用蛋白提取移液器处理。这些数据表明，从每个样品的分离蛋白是免疫反应，从而引发了积极的回应。 表明，分离蛋白分数是没有污染的核酸，蛋白质样品进行PCR扩增（如例1中所述）。这些实验的结果均为阴性凝胶电泳。因此，我们得出这样的结论：样品的蛋白质含量污染核酸分离。这些结果，在与核酸分离步骤表明，核酸和蛋白质含量分离在不同的分馏步骤中描述的整体。 T“&gt;例3：分离蛋白是适合电泳10 图5描述了1考马斯亮蓝染色8％丙烯酰胺凝胶从6M胍异硫氰酸使用吸管和相关提取分离与牛血清白蛋白（ 图5，通道A和C）和BSA加载协议。分离蛋白的电泳迁移率是控制样品相同，因此，我们得出这样的结论：提取过程中不改变分离蛋白的共价结构。 <img alt="图1a" src="/files/ftp_upload/4202/4202fig1A.jpg" /> 图1中，A组：混合样品在样品管与6M胍硫氰酸和吸附剂五倍通过描写一个典型的核酸提取过程。最后一段后，样品被加入蛋白提取液。吸附剂和结合的核酸用乙醇洗两次。样品是空气ð里德和吸附剂洗脱。 <img alt="图1b" src="/files/ftp_upload/4202/4202fig1B.jpg" /> 图1，面板A：一个典型的蛋白提取过程的描写，从步骤1， 图1中 ，委员会第二次提取移液器15倍以上的吸附剂通过与蛋白质隔离缓冲混合样品。该过程的其余部分是相同的核酸提取协议。 <img alt="图2" src="/files/ftp_upload/4202/4202fig2.jpg" /> 图2：溴化乙锭染色的琼脂糖凝胶负图片一个复用的志贺毒素1和2基因扩增实验，进行文化样本（通道A和B）或未经处理的污水样品添加与E.大肠杆菌 O157：H7型（车道D，E和F）的。样品被直接添加到PCR反应管（通道A和D）或样品进行了处理使用的隔离议事录和核酸提取到PCR反应管（样品B，E和F）。 PCR的协议是由丰等 11描述的修改。我们的修改只针对那些STX 1（低频段）和STX 2（上带）对引物扩增，所有其他的引物对辍学。巷C是1KB梯子从BioRad公司。 <img alt="图3" src="/files/ftp_upload/4202/4202fig3.jpg" /> 图3：E.紫外光谱结果大肠杆菌 O157：H7型核酸分离使用的报告提取移液器孤立核酸标记隔离A和隔离B。样品的紫外光谱测试之前，核酸提取和标记前隔离。光谱获得使用日立U3010分光光度计和相关的软件包。 <img alt="图4" src="/files/ftp_upload/4202/4202fig4.jpg" /><br/&gt; 图4：彩色照片的RapidChek试纸图2中使用的样品的蛋白质含量分离提取移液器和横向流动检测免疫反应的测试。车道交流未处理的控制，细胞培养，加入无核酸或蛋白质提取试纸。弄一个是E。大肠杆菌 BL21 DE3 pLysS中作为阴性对照，泳道B和C显示大肠杆菌大肠杆菌 O157：H7的是未经处理的（B），或与丝裂霉素C（C）4小时处理。通过H车道ð显示提取的蛋白，以增加横向流动试纸的结果。车道D和E分别探讨了利用大肠杆菌蛋白提取物要么没有（D）或（E）为阴性对照丝裂霉素C治疗大肠杆菌 BL21 DE3 pLysS中。车道跳频结果显示，当从大肠杆菌中提取蛋白大肠杆菌 O157：H7使用。蛋白提取未经处理的（F），或从2小时（G）或4小时（H）丝裂霉素C处理文化。阳性结果产生双红线。 <img alt="图5" src="/files/ftp_upload/4202/4202fig5.jpg" /> 图5：照片的考马斯亮蓝染色的SDS-PAGE BSA的分离提取移液器和描述协议的解决方案。车道A和C是250微克/毫升和500微克/毫升，分别控制车道。 B和D车道描绘康复牛血清白蛋白，从含有相同浓度为各自控制的解决方案。

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Electricity-Free, Sequential Nucleic Acid and Protein Isolation

工具和化学描述顺序隔离没有对电力的需要，由蛋白质样品的核酸。该工具由吸附剂而隔离化学固相萃取原则的基础上，移液管内举行。孤立的大分子可以基于免疫和基于PCR的检测分析。

免电力，顺序的核酸和蛋白质的分离

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

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JoVE Journal

Biology

12.4K 次观看.  CUBRC, Inc.. 工具和化学描述顺序隔离没有对电力的需要，由蛋白质样品的核酸。该工具由吸附剂而隔离化学固相萃取原则的基础上，移液管内举行。孤立的大分子可以基于免疫和基于PCR的检测分析。

视频: Electricity-Free, Sequential Nucleic Acid and Protein Isolation

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

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 ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

核酸结构分析及建设3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

从发展胚胎小鼠心脏瓣膜区蛋白分离

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

高效的核酸提取和16S rRNA基因序列的细菌群落特征

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...