1.抗体捕获到高容量磁蛋白A或磁G蛋白珠<ol><li>均匀再通过暂停轻轻摇动磁珠。保持均匀的悬浮液珠等分的时候。 </li><li>加入50μl珠浆料到1.5ml微量离心管中。在磁力架，持续10秒的地方。小心取出存储缓冲区。 </li><li>加入250微升抗体结合缓冲。 </li><li>混合并在磁性支架10秒的地方。小心取出结合缓冲液。 </li><li> ，增加1.0毫升含50-100微克抗体与珠粒样品。样品可被纯化在细胞培养基的抗体或抗体。 </li><li>使用涡旋混合器或最终过端混合器在RT混合样品60分钟。 </li><li>放置管在磁性支架10秒并除去上清液。 </li><li>加入250微升抗体结合/洗涤液混合。在磁性支架放置10秒，取出结合/洗涤缓冲液。重复这个STEP总共两次洗涤的。 </li><li>继续第2节使用胺化学或第3节使用硫醇化学标签抗体标签抗体。 </li></ol> 2.抗体标记使用胺化学<ol><li>共轭抗体<ol><li>添加100μl的胺偶联缓冲液中的珠。 </li><li>通过加入100微升二甲基亚砜（DMSO）中的至1.0mg染料溶解在10毫克/毫升的胺反应性的荧光染料（AlexaFluor 532-SE）。震荡混合。使该解决方案使用前。 </li><li>添加2.5微升的胺 - 反应性染料为100微克的抗体。 注意:通常建议一个5-20摩尔过量活性染料。然而，反应性染料的量加入到反应需要根据所需染料与抗体比例和特性的抗体性质来凭经验优化。 </li><li>使用涡旋混合器或最终过端混合器在RT混合样品60分钟。确保珠留在悬索桥ñ。 </li><li>放置管在磁性支架10秒并除去上清液。 </li><li>加入250微升抗体结合/洗涤液混合。在磁力架，持续10秒的地方。取下并丢弃绑定/洗涤液。重复此步骤，总共两次洗涤。 </li></ol></li><li>抗体恢复<ol><li>加入50μl洗脱缓冲液至珠。 </li><li>使用涡旋混合器或最终过端混合器在RT混合5分钟。确保珠粒留在悬浮液中。 </li><li>放置管在磁性支架10秒。除去洗脱样品，并转移到含有5微升中和缓冲液的一个新的微离心管中。 </li><li>重复这个过程更多的时间，集中洗脱的样品。 </li><li>定量抗体浓度，并如第4所述的染料与抗体的比例。 </li></ol></li></ol> 3.抗体标记使用硫醇化学<ol><li>减少抗体<ol><li>加入250微升巯基结合缓冲和组合。放置管在磁性支架10秒。取下并丢弃缓冲区。重复此步骤两次。 </li><li>加入100微升巯基结合缓冲。 </li><li>添加二硫苏糖醇（DTT）至2.5mM的终浓度。 </li><li>使用涡旋混合器或最终过端混合器在RT混合样品60分钟。确保珠粒留在悬浮液中。 </li><li>放置管在磁性支架10秒，并丢弃缓冲器。 </li><li>加入250微升巯基结合缓冲液混匀。放置管在磁性支架10秒。取下并丢弃缓冲区。重复此步骤，总共两次洗涤。 </li><li>加入100微升巯基结合缓冲。 </li></ol></li><li>共轭抗体<ol><li>通过加入100微升的DMSO至1.0mg染料溶解在10毫克/毫升的硫醇反应性染料。震荡混合。使该解决方案使用前。 </li><li>添加2.5微升硫醇反应性染料为100微克抗身体。 注意:通常建议一个5-20摩尔过量活性染料。然而，反应性染料的量加入到反应需要根据所需染料与抗体比例和特性的抗体性质来凭经验优化。 </li><li>使用涡旋混合器或最终过端混合器在RT混合样品60分钟。确保珠粒留在悬浮液中。 </li><li>把试管磁力架，持续10秒，取出上清。 </li><li>加入250微升巯基结合缓冲液混匀。在磁保持10秒的地方，去掉缓冲区。重复此步骤，总共两次洗涤。 </li></ol></li><li>洗脱抗体<ol><li>加入50μl洗脱缓冲液至珠。 </li><li>使用涡旋混合器或最终过端混合器在RT混合5分钟。确保珠粒留在悬浮液中。 </li><li>放置管在磁性支架10秒。删除洗脱样品，并转移到含有5微升NEUT的一个新的微离心管ralization缓冲区。 </li><li>重复这个过程更多的时间，集中洗脱的样品。 </li><li>定量抗体浓度，并如第4所述的染料与抗体的比例。 </li></ol></li></ol> 4.计算染料对抗体的比率<ol><li>测量抗体 - 染料偶联物的280nm处的吸光度（A280），并在λ最大为染料（安迈）。 </li><li>计算抗体浓度: 抗体浓度（毫克/毫升）= A280 - （A× 最大 CF）/ 1.4 其中，CF染料（由生产商提供的）的=校正因子。 </li><li>计算染料与抗体的比例: 染料与抗体的比率（DAR）=（A 最大值 ×15万条）/抗体浓度（毫克/毫升）×ε 染料 式中，ε 染料 =在其最大吸光度的染料的消光系数和抗体的分子量= 150000道尔顿。 </li></ol>

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Authors are employees of Promega Corporation.

本研究的目的是开发标记抗体中，存在于在低浓度的细胞培养基，具有多种小分子的方法。这种方法将允许大量的抗体，在抗体发现的早期阶段，与小分子进行标记，并使用生物相关测定法进行筛选。一种这样的测定法是受体介导的抗体的内化测定法，其中内化可能甚至具有相似的结合亲和力的抗体之间变化。因此，为了具体地筛选抗体除了传统的酶联基于免疫吸附测定（ELISA）筛选其内在特性是重要的。在早期的抗体开发阶段来标记抗体方法的主要要求是:a）标记直接从细胞培养基抗体的能力; b）向标记以低浓度存在的抗体，例如，当在细胞培养基中的抗体浓度为50微克/毫升的能力; C）标记的抗体和抗体浓度的纯度应当与下游应用兼容; d）该方法应与胺和硫醇偶联化学相容; e）该方法应该与同时处理多个样品相容。 考虑到这些需求，我们开发了采用高容量磁蛋白A和G蛋白珠的小球上的抗体标记和纯化方法。这些珠粒基于具有低的非特异性结合性的亲水性纤维素和导致高纯度的抗体制剂， 如图2珠的高容量意味着珠少量可有效地捕获抗体从媒体和抗体可在小体积，因此浓缩抗体溶出。在表1中 ，只有10微升结算珠粒用来捕捉由1.0毫升样品抗体（预期浓度50-100微克/毫升）的ð标记的抗体在200-500微克/毫升之间的浓度为120微升体积被洗脱。这些浓度与基于细胞内化实验，其中典型的抗体浓度范围是从1.0微克/毫升至10毫微克/毫升15,16兼容。上的珠标记方法是与两个胺和硫醇化学和广泛的染料与抗体的比率可以与标记的抗体（ 表1）的有效回收沿来实现兼容。实验均一式三份进行，并多达12个样品在平行人工处理，显著减少处理时间。标记的吞吐量可进一步增加通过使用机器人平台，因为它已经在使用磁珠19,22的其他应用程序被显示。 具有较高的抗体量的样品可以通过增加珠粒用于捕获，同时保持抗体的回收和标记efficie的量来容易地适应NCY（ 见表2）。该方法也与多种染料兼容，使得各种染料标记抗体可制成，然而，染料与抗体的比率将需要进行优化。染料2-4抗体比率被确定为是最佳的11,12和已批准的ADC的情况下的3.5药物每个抗体的平均已经报道23。用小球上的缀合化学，它是比较容易调取决于下游应用的要求每个抗体的小分子的最优数目。此外，既为标签硫醇和胺化学可以进行测试，以确定最佳的化学选择，因为它已经表明，抗原和受体结合， 在体内稳定性是用于ADC的关键的，和抗体的治疗活性依赖于结合化学24 25。 有以达到良好的抗体标记和恢复的一些关键因素。小分子的性质重新两种抗体的回收和染料与抗体的比例的临界驱动力。对于疏水性小分子，高染料与抗体的比例将引起标记的抗体的珠的非特异性粘附和显著还原的抗体恢复。有时，这取决于染料和抗体的特性，我们发现，一个小珠可能比另一种更好，因此，在优化过程中的两个小珠的检测可能是有用的。蛋白A和蛋白G对和不同的亚型，其中应选择适当的珠时，应考虑的来自不同物种的抗体不同的亲和力21。例如，A蛋白，因此有G蛋白珠强烈建议小鼠IgG1低亲和力。对于硫醇化学，DTT和TCEP还原剂同样出色的工作。 DTT将与马来酰亚胺反应，因此应当通过洗涤完全除去然而干扰。 2.7低pH推荐用于洗脱，但可能需要用于有效REC 2.2较低的pH卵巢中在某些情况下。对于可能在低pH变性标记的抗体有效地回收和抗体的功能之间的平衡将不得不凭经验确定。它公知的是化学标记可能影响抗体-抗原结合活性11,23，因此，这是绝对必要使用所需的下游应用标记后测试对于抗体功能。 总之，我们描述标签直接从细胞培养基的荧光染料的抗体没有任何事先纯化步骤的方法。这种方法利用了高容量磁性蛋白A和蛋白G磁珠和超过基于溶液标记方法的显著改善需要高度纯化的抗体在高浓度10,11。使用磁珠允许手动和自动样品处理，这是通过基于非磁性珠标记技术17,18的优点。最后，使用高容量的B​​EADS允许抗体标记反应的规模从低微克的水平数百微克的使用少量的珠子和区别于其他磁珠基于标记方法19此方法。这里介绍的协议描述的抗体的标记用荧光染料，但该方法可以扩展到其它的标签包括生物素，细胞毒性药物和可能的酶。

标记的小分子抗体是也许是生物学1,2-最常用的试剂。用荧光染料标记和生物素的抗体在成像，免疫测定被广泛使用，流式细胞术，Western印迹，免疫沉淀和其他应用程序3-6中。放射性标记的抗体3,7-发现在成像和治疗，标有细胞毒性药物（ADC）的抗体广泛使用用于癌症的治疗中，提供新的选择，和两个ADC已经被批准用于治疗用途8。尽管他们广泛使用，用于标记抗体的方法仍然令人惊讶的改变，通常涉及多个反应和脱盐步骤9-12。溶液的方法中，其中只有少数的抗体需要被标记，并在高度纯化的形式提供在高浓度情况下，并且在足以卷工作得很好。然而，对于象ADC的新的应用程序，还有一个需要在早期杂交瘤细胞阶段来标记抗体，使它们可以筛选生物相关的属性，例如，受体介导的抗体的内化13-16。在杂交瘤阶段，样品体积是有限的，抗体在低浓度下表达和若干样本都很大，因此在基于溶液的标记方法是不适合的。 简化和改进了传统的抗体标记方法的吞吐量，一些替代办法，已经提出17,18。一种方法是使用非磁性蛋白装在小的列A亲和珠捕获抗体随后标记反应和标记的和纯化的蛋白的洗脱。这种方法可用于直接从细胞培养基标记的抗体，然而，使用列可以是费力。磁珠基于方法最近有报道19，消除了使用列和提高吞吐量但由于在有限的抗体结合的珠容量，仅纳克至抗体的低微克量可以进行标记。 我们最近开发并用于高容量磁性蛋白A和蛋白G珠（&gt; 20毫克人IgG /毫升沉降的珠子）标记存在于细胞培养基与小分子20的抗体。珠的高容量允许几十到几百抗体微克能够方便标记和珠的快速磁响应简化的大量平行样品的处理和加工。使用荧光染料作为代理人为小分子，我们表明，该方法是与胺和硫醇化学标记兼容，并提供了标记，并非常纯净抗体的高回收率。 这个协议和所附视频描述存在于使用磁性蛋白A和蛋白G珠的细胞培养基小鼠抗体的小球上的标记。该协议是分为四个部分:第一部分介绍到生物样品珠抗体捕获。继拍摄，采用胺化学或使用硫醇化学与荧光染料抗体的标签在第2和第3节分别描述。最后，第4描述了一种用于抗体浓度的计算和染料与抗体的比例的方法。

<table><tbody><tr><td>Magne Protein A Beads</td><td>Promega Corporation</td><td>&amp;nbsp;G8781</td><td></td></tr><tr><td>Magne Protein G Beads</td><td>Promega Corporation&amp;nbsp;</td><td>&amp;nbsp;G7471</td><td></td></tr><tr><td>AlexaFluor 532-SE (Succinimidyl Ester)</td><td>Life Technologies</td><td>A20001</td><td></td></tr><tr><td>AlexaFluor 532-ME (Malemide)</td><td>Life Technologies</td><td>A10255</td><td></td></tr><tr><td>AlexaFluor 647-ME (Maleimide)</td><td>Life Technologies</td><td>A20347</td><td></td></tr><tr><td>Fluorescein-ME (Maleimide)</td><td>Life Technologies</td><td>F-150</td><td></td></tr><tr><td>Magnetic Stand</td><td>Promega</td><td>Z5332</td><td></td></tr></tbody></table>

antibody labeling with fluorescent dyes using magnetic protein a and protein g beads

标有类似的荧光染料，细胞毒性药物和放射性示踪剂的小分子抗体是生物医学研究的重要工具，免疫诊断试剂以及最近作为治疗剂。对于小分子标记抗体的传统方法需要在相对高浓度的纯化抗体，涉及多个透析步骤和有限的吞吐量。然而，一些应用，包括抗体药物偶联物（ADC）的领域中，将可使用的新方法，这将允许抗体的标记直接从细胞培养基。这样的方法可以允许抗体在生物学相关测定法进行筛选，例如，在ADC的情况下，受体介导的抗体内测定。这里，我们描述的方法（小球上的方法），使少量的抗体的标记直接从细胞培养基。这种方法利用高容量磁蛋白A和G蛋白亲和珠捕获抗体从细胞培养基，随后通过标签使用任一胺或硫醇化学和标记抗体的随后洗脱的小分子。以荧光染料作为替代物的小分子，我们展示了三种不同的小鼠抗体的小球上的标签直接从同时使用胺和硫醇化学标记细胞培养基。抗体与蛋白A和蛋白G的高结合亲和力，确保高回收率，以及标记的抗体的纯度高。另外，使用的磁珠允许多个样品进行人工处理，从而显著提高标签的吞吐量。

一种使用高容量磁性蛋白A和蛋白G磁珠标记的小分子的抗体概略示于图1。抗体上的磁性蛋白G珠可与小分子标记捕获，例如荧光染料，即使用胺化学哪些标签赖氨酸氨基酸或用硫醇化学哪些标签在抗体的铰链区还原硫醇的伯胺。抗体和蛋白G之间的亲和力强21和结果在标记反应中抗体的损失最小。相比简单的纯化时，这反映在与荧光染料标记后三种不同的小鼠抗体的同种型（ 表1）的有效回收（50-90％）。有效地回收（类似于19之前报告的）也很明显在考马斯凝胶（ 图2），其中带的Intensi从纯化的抗体的重和轻链的关系和标记抗体反映在表1中所报告的结果。磁性蛋白G珠具有产生高度纯化的抗体的低非特异性结合性质如在考马斯凝胶（ 图2）看到的和既重和轻链的荧光标记（ 图2）。小球上的标签，也可以在磁性蛋白A珠完成并与导致高的回收率和良好的染料与抗体的比率（ 表2）的多个荧光染料兼容。 <img alt="图1" src="/files/ftp_upload/54545/54545fig1.jpg" /> 图1.原理与荧光分子抗体的小球上的标签。 使用硫醇化学（A）抗体标记。抗体是在高容量磁蛋白A或磁蛋白G B抓获接着还原的使用如DTT或三（2-羧乙基）膦（TCEP）的还原剂链间二硫键EADS。含有荧光染料硫醇反应性的马来酰亚胺添加到标签的抗体。标记的抗体是由低pH洗脱回收并立即中和。使用胺化学（B）抗体标记。关于高容量磁性蛋白A和蛋白G珠子捕获抗体与胺反应性的荧光染料反应在赖氨酸的伯胺来标记抗体。标记的抗体是由低pH洗脱洗脱，并立即中和。从纳特，N。等20改编<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54545/54545fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54545/54545fig2.jpg" /> 图2. 变性小号三次鼠标的IgG的DS-PAGE凝胶上的图像同种型的纯化和用AlexaFluor 532标记的，使用小球上的方法。（A）的胺反应（B）的硫醇反应。标有硫醇反应性染料的抗体的凝胶图像。 1-6道对应于表1所示的样品。将凝胶用荧光扫描仪第一成像并只显示AlexaFluor 532标记的抗体重和轻链。样品，其中抗体只纯化是不可见的。凝胶随后用考马斯染料染色以显示从该纯化以及被标记的抗体的抗体重链和轻链。从纳特，N。等 20改编<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54545/54545fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td></td&gt; <td></td><td colspan="2"> 胺反应 </td><td colspan="2"> 巯基反应 </td></tr><tr><td> 好 </td><td> 同型 </td><td></td><td> 抗体恢复（微克） </td><td> 染料对抗体的比率 </td><td> 抗体恢复（微克） </td><td> 染料对抗体的比率 </td></tr><tr><td> 1 </td><td rowspan="2">小鼠IgG1 </td><td>纯化</td><td> 54.8±1.8 </td><td> 0 </td><td> 49.2±2.0 </td><td> 0 </td></tr><tr><td> 2 </td><td>共轭</td><td> 27.4±3.2 </td><td> 4.2±0.2 </td><td> 46.​​2±3.5 </td><td> 1.6±0.2 </td></tr><tr><td> 3 </td><td rowspan="2">小鼠IgG2a </td><td>纯化</td><td> 85.9±6.4 </td><td> 0 </td><td> 75.3±8.0 </td><td> 0 </td></tr><tr><td> 4 </td><td> Conjug通货膨胀</td><td> 60.7±3.2 </td><td> 4.4±0.1 </td><td> 61.5±7.4 </td><td> 5.5±0.4 </td></tr><tr><td>五</td><td rowspan="2">小鼠IgG2b </td><td>纯化</td><td> 79.9±6.9 </td><td> 0 </td><td> 73.3±1.4 </td><td> 0 </td></tr><tr><td> 6 </td><td>共轭</td><td> 66.7±0.5 </td><td> 5.6±0.1 </td><td> 55.7±0.9 </td><td> 5.2±0.5 </td></tr></tbody></table> 表1:直接从细胞培养基使用胺和硫醇化学三次鼠标IgG同种型在珠标记对于每个样品， 使用只是用磁性蛋白G珠（50μl的浆液）和第二等分试样用于小球上的标记与胺 - 反应性AlexaFluor 532染料的抗体的纯化1.0毫升细胞培养基中的一个等分试样。在这两种情况下抗体的回收率计算（如在文本中所描述）的ð比较，以确定标记反应过程中抗体的损失。染料与抗体的比例也被计算所有三种抗体。所有反应一式三份进行。从纳特，N。 等调整。20 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td></td><td colspan="2"> 磁G蛋白珠 </td><td colspan="2"> 磁蛋白A珠 </td></tr><tr><td></td><td></td><td> 抗体恢复（微克） </td><td> 染料对抗体的比率 </td><td> 抗体恢复（微克） </td><td> 染料对抗体的比率 </td></tr><tr><td> 1 </td><td>纯化</td><td> 263.7±10.4 </td><td> 0 </td><td> 269​​.4±5.1 </td><td> 0 </td></tr><tr><td> 2 </td><td> AlexaFluor532 </td><td> 182.9±15.3 </td><td> 5.377; 0.04 </td><td> 189.1±6.9 </td><td> 6.9±0.2 </td></tr><tr><td> 3 </td><td> AlexaFluor647 </td><td> 192.5±2.9 </td><td> 3.3±0.1 </td><td> 112.3±11.4 </td><td> 3.6±0.2 </td></tr><tr><td> 4 </td><td>荧光素</td><td> 179.5±5.3 </td><td> 6.8±0.1 </td><td> 201.5±6.5 </td><td> 6.8±0.1 </td></tr></tbody></table> 表2:在珠标记三种不同硫醇反应性荧光染料小鼠IgG2a的浓缩细胞培养基样品在本实验中使用，以显示该方法也可用于不同量抗体的通过。此外，使用磁蛋白G以及磁蛋白A的一个为1.0ml使用磁性蛋白G珠（100微升浆料）浓缩细胞培养基用于抗体纯化等分试样进行缀合反应。其他三个1.0毫升等分小号进行标记，并与硫醇反应性AlexaFluor 532，AlexaFluor 647和荧光素纯化。计算抗体回收率（如在文本中所描述）和用于各种标记反应进行比较。染料与抗体的比例，并计算所有三个荧光染料。类似的净化和标签使用磁蛋白A珠完成。所有反应一式三份进行。从纳特，N 等人改编"20。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 缓冲 </td><td> 评论/说明 </td></tr><tr><td>胺偶联缓冲液（10mM碳酸氢钠缓冲液，pH 8.5） </td><td> 1.0.084克碳酸氢钠 2.在去离子水中溶解。调节至pH 8.5。 3.调整最终体积至100毫升去离子水。 </td></tr><tr><td>硫醇偶联缓冲液（10用1mM EDTA，pH为7 mM磷酸盐缓冲液。0） </td><td> 1.0.0378克磷酸钠，二氢，一水合物 2.0.195克磷酸钠，二元，七水合物 3.在去离子水中溶解。 4.加入EDTA至1.0mM的终浓度。 5.调整至pH 7。 6.调整最终体积至100毫升去离子水。 </td></tr><tr><td>抗体结合/洗涤缓冲液（10mM磷酸盐缓冲液，pH 7.0） </td><td> 1.0.0378克磷酸钠，二氢，一水合物 2.0.195克磷酸钠，二元，七水合物 3.在去离子水中溶解。调节pH至7。 4.调整最终体积至100毫升去离子水。 </td></tr><tr><td>洗脱缓冲液（50mM甘氨酸缓冲液，pH值2.7） </td><td> 1.0.188克甘氨酸 2.在去离子水中溶解。调节pH至2.7，用HCl。 3.调整最终体积至50ml去离子水。 </td></tr><tr><td>中和缓冲液（2M的Tris缓冲液，pH7.5）中</td><td> 1.0.472克TRIZMA基地 2.2.54克TRIZMA盐酸盐 3.在去离子水中溶解。调节pH至7.5。 4.调整最终体积至10ml去离子水。 </td></tr></tbody></table> 表3:缓冲组合物。

Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

上的胎圈的方法用于标记抗体与小分子使抗体的少量的标记直接从细胞培养基。这种方法是与胺和硫醇化学相容，并且可以并行处理多个样品，手工或使用自动平台。

抗体标记用荧光染料使用磁蛋白A和G蛋白珠

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

antibody-labeling-with-fluorescent-dyes-using-magnetic-protein

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.8K 次观看.  Promega Corporation. 上的胎圈的方法用于标记抗体与小分子使抗体的少量的标记直接从细胞培养基。这种方法是与胺和硫醇化学相容，并且可以并行处理多个样品，手工或使用自动平台。 

视频: Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

用于检测实验方案<em&gt;蓝藻</em&gt;与抗体基因芯片液体和固体样品

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

科研

环境科学

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

神经科学

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

生物化学

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

生物学

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

具有邻近标记蛋白质组学的神经元溶酶体相互作用组表征

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

在联合电子能谱成像miniSOG标记的DNA修复蛋白可视化（ESI）

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

小鼠肝脏中细胞类型特异性基因表达分析

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

使用荧光相关光谱检测蛋白质聚合

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins

Source: Aishwarya, R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64085">Visualizing Subcellular Localization of a Protein in the Heart Using Quantum Dots-Mediated Immuno-Labeling Followed by Transmission Electron Microscopy.</a> J. Vis. Exp. (2022).
This video demonstrates a technique for the immunolabeling of sigma-1 receptors in a murine heart tissue section using quantum dots. The heart tissue is fixed, osmicated, stained, dehydrated, embedded in resin, and then sectioned. The sections are then treated with antibodies and labeled with quantum dots to demarcate the subcellular localization of Sigma-1 receptors using electron microscopy.

一种可视化蛋白质亚细胞定位的免疫标记技术

1. Animal preparation

	Anesthetize the mice using 3% isoflurane. Open the chest by making a horizontal incision in the region above the middle abdomen ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

Source: Blanco, Y. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54994">Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strains through fluorescence in distinct spots.

抗体标记用荧光染料使用磁蛋白A和G蛋白珠

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用于检测实验方案