Name: antibody transfection into neurons as a tool to study disease pathogenesis
Uploaded: 2012-09-26T13:00:00
Description: Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

这项工作是支持的研究与发展，医学研究服务，退伍军人事务部办公室。这项研究是由一个VA优秀评论奖（MCL）大学和田纳西州健康科学中心多发性硬化症研究基金。

1。抗体标记（图1） <ol><li> 0.1 M NaHCO3缓冲： <ul type="circle"><li> 8.4克的NaHCO 3 </li><li> 29.2克氯化钠</li><li> 1升H 2 O </li></ul></li><li>带来的缓冲与此溶液的pH为8.4（10.6克Na 2 CO 3，29.2克NaCl，1升H 2 O）。 </li><li>加入35微升Atto550 NHS到第70微升抗hnRNPA1和500μl的NaHCO 3缓冲液，并在室温下的1小时旋转。长达一小时的旋转后，将混合物注入到一个透析器，?...

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为了测试假设特定的分子和它们的表达，基因和其他载体转染到细胞中。此过程中的独特的方面是能够容易地转染到靶细胞的抗体。我们进行了这种方法在5个不同的实验中，各一式两份。在所有的实验中，转染效率的结果是相似的。更重要的是，该方法使进入细胞的抗体的条目而不改变抗体的结构或功能。另外，用荧光显微镜的活神经元的使用，我们能够确定，通常超过55％的转染效率。这允许?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>荧光显微镜AxioObserver A1 </td><td>蔡司</td><td></td></tr><tr><td> Axiovision 4.2版</td><td>蔡司</td><td></td></tr><tr><td>反rhnRNPA1 </td><td> Abcam公司</td><td> Ab4791 </td></tr><tr><td> FITC标记的抗RIGG </td><td> OZ生物科技公司</td><td> AI20100 </td></tr><tr><td> Atto550 NHS酯</td><td>西格玛</td><td> 92835 </td></tr><tr><td> AB-DeliverIN的</td><td> OZ生物科技公司</td><td> AI20100 </td></tr><tr><td> DMEM/F12 +10％FBS +1％抗生素</td><td> Gibco公司</td><td> 11320-033 </td></tr><tr><td>聚-D-赖氨酸的8孔文化相片数</td><td> BD </td><td> 354632 </td></tr><tr><td>多功能肩</td><td>科技INDUSTRIES公司</td><td>型号151 </td></tr><tr><td> DAPI安装介质</td><td>密理博</td><td> S7113 </td></tr><tr><td> PBS </td><td> Ambion公司</td><td> 9626 </td></tr><tr><td>透析</td><td>赛默飞世尔科技</td><td> 66380 </td></tr><tr><td>的Amicon Ultra-0.5 </td><td>密理博</td><td> UFC501096 </td></tr><tr><td>多聚甲醛（4％溶液） </td><td>电子显微镜科学</td><td></td></tr><tr><td>纳米掉落</td><td>赛默飞世尔科技</td><td></td></tr></tbody></table>

antibody transfection into neurons as a tool to study disease pathogenesis

抗体的能力，获得新的洞察发生在生命系统的各种事件。产生非常具体的目标蛋白的抗体，可以非常精确的生物学问题的能力加以解决。更重要的是，抗体的发病机制中的数量的人类疾病包括系统性红斑狼疮（SLE），类风湿性关节炎（RA），副肿瘤综合征，多发性硬化症（MS）和人T-淋巴病毒1型（HTLV-1）有牵连相关性脊髓病/热带痉挛性瘫痪（HAM / TSP）1-9。如何抗体，引起病是一种正在进行的调查，数据表明，抗体和各种细胞内的分子在炎症之间的相互作用，改变细胞的信息传递，细胞凋亡10。它已被证明，MS和HAM / TSP患者产生自身抗体，细胞内的RNA结合蛋白异构核糖核酸蛋白甲1（中hnRNP A1）3，5-7，9，11。最近的数据表明，内神经元和表面抗原的抗体是致病3，5-9，11。因此，一个程序，允许细胞内抗体的研究蛋白质相互作用，将给予很大的洞察疾病的发病机制。 基因通常在培养的原代细胞和细胞系转染，但转染进入细胞的抗体已被阻碍，通过改变抗体的结构或转染效率差12。其它转染方法包括基于阳离子脂质体（由DOTAP / DOPE）和聚乙烯亚胺（PEI）的抗体的转染，这两种抗体转染在10倍的减少相比，控制12。在我们的研究中进行的方法是类似的阳离子脂质介导的方法，并使用的脂质为基础的机制，以形成非共价复合物与抗体通过静电和hydrophobic的相互作用13。我们利用的Ab-DeliverIN试剂，这是能够捕获抗体通过非共价键的静电和疏水相互作用，并提供它们在细胞内的脂质制剂。因此，化学和遗传联轴器交付到活细胞的功能性抗体是没有必要的。此方法使我们能够执行各种抗体跟踪和蛋白定位的实验，以及作为细胞内抗体的分子后果：蛋白质相互作用9分析。 在这个协议中，我们将展示如何为神经元转染抗体快速，重复性好，具有高度的转染效率。作为一个例子，我们将使用抗核糖核蛋白A1和抗IgG抗体。对于容易量化的转染效率，我们使用抗核糖核蛋白A1与ATTO-550-NHS和FITC标记的IgG抗体标记。 Atto550 NHS是一个新的标签与的高分子吸收和量子易领域。激发源和荧光过滤器Atto550是类似的Cy3标记（例如：556 EM 578）。 Atto550此外，具有高的耐光性。 FITC标记的IgG作为对照表明，该方法是多才多艺的，不染依赖。这种方法产生的数据将有助于了解人类疾病的发病机制中可能发挥的作用，细胞内的靶抗原的抗体。

Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

一种快速的方法，调查相关的抗体的存在下，在细胞内环境中的分子机制的相互作用和效果进行说明。该方法涉及的抗体的转染到活细胞，使用的基础上的脂质制剂的非共价复合物的形成。该技术是适应的永生化细胞系和原代细胞。

抗体转染神经元作为一种工具来研究疾病的发病机制

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Research

JoVE Journal

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神经科学

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