Name: antibody labeling with fluorescent dyes using magnetic protein a and protein g beads
Uploaded: 2016-09-15T14:00:00
Description: Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

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...

<ol>
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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?...

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 border="0" cellpadding="0" cellspacing="0" width="592">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Magne Protein A Beads</td>
			<td style="width:103px;">Promega Corporation</td>
			<td style="width:119px;">&#160;G8781</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Magne Protein G Beads</td>
			<td style="width:103px;">Promega Corporation&#160;</td>
			<td style="width:119px;">&#160;G7471</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 532-SE (Succinimidyl Ester)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A20001</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 532-ME (Malemide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A10255</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 647-ME (Maleimide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A20347</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluorescein-ME (Maleimide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">F-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic Stand</td>
			<td style="width:103px;">Promega</td>
			<td style="width:119px;">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?...

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, ...

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