Name: consensus brain-derived protein, extraction protocol for the study of human and murine brain proteome using both 2d-dige and mini 2de immunoblotting
Uploaded: 2014-04-10T15:30:00
Description: Watch this Scientific Journal Video about Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting at JoVE.c

这项工作是由INSERM的，里尔2大学的支持，MEDIALZ，LabEx（卓越实验室，计划为未来投资）和DISTALZ（创新策略的一个跨学科的方法来老年痴呆症的发展）。 FJ.FG目前的ANR（法国国家研究署/ NeuroSplice去头PROJET ANR-2010-BLAN-1114 01）收件人奖学金，但这项工作也是下授出从JCCM（西班牙）的支持。

1，同质化，并从人类和小鼠脑组织总蛋白抽提<ol><li>脑组织的同质化。均匀化用陶工玻璃（用于人类样品），或特氟隆匀浆器（用于小鼠的样品）的脑组织中的10％（重量/体积）的8M尿素，2M硫脲和1％w / v的SDS缓冲液（UTS）。超声在60 Hz 30脉冲申请0.5秒，共组织解体13，14一个超声波发生器。 <ol><li>测定蛋白浓度与Bradford法，以BSA作为标准。这UTS缓冲器提取是用一维SDS-PAGE上完全兼容，只要...

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+fluorescence+difference+gel+electrophoresis+for+comparative+proteomics+profiling.">Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling.</a> Nat Protoc. 1, 1732-1742 (2006).</li></ol>

发现蛋白质的表达病理变化，寻找生物标记物和药物靶潜在途径的调节是其中neuroproteomics的目标接近30。在一片新兴的工具，在二维超声心动图场增加了一个有前途的预想。然而，一个共识应以尽量减少可变性和提高重现性在实验中达到。在这种思想的行标准化协议来执行2D-DIGE（ 图2）和随后的2DE免疫印迹（ 图3），对人类和小鼠的脑组织与一维免疫印迹完全兼容...

精神和神经紊乱代表全球疾病负担的13％，如病理生理机制，危险因素和前驱的生物标志物的新挑战，必须探索1。为配合这一目标，人类大脑的蛋白质组学研究成为不可或缺揭开参与像记忆，行为，情绪和神经元的可塑性，例如，不仅对生理也为病理状态过程的分子途径。因此，在使用动物模型的更具体的转基因小鼠，带来了广泛的可能性，以模仿人类神经退行性疾病2的病因。 蛋白质组学方法是时下为了实现这些新的观点在神经科学领域。二维凝胶电泳（2DE）是一种有效的和可能的简单的方法，使比较大范围的样品的蛋白质组。此外，它也是一个强大的方法，以便确定从复杂混合物中分离出的蛋白质和通过质谱法进一步分析。这种技术基本上是由在两个连续的步骤：根据其等电点（pI）1）蛋白的分离通过等电聚焦（IEF）。更精确地说，将电势穿过固丙烯酰胺条施加之间的pH梯度，然后将蛋白质转移和集中在其全球净电荷的函数所确定的等电点。 2）等电聚焦蛋白质变性并通过加入十二烷基硫酸钠（SDS带负电荷），从而在其第一结构的蛋白质根据它们的表观分子量（MW）用SDS-PAGE 3分离。这两个不同的特性使我们能够解决一个double值在蛋白质组研究中走得更远。一方面这种方法提供了可能用最小的染料2D-DIGE法进行定量分析，并在另一方面一个qualitativE解析由微型二维超声耦合到西方墨点法。 通过2D-DIGE定量分析赐蛋白表达的变化遍布样品蛋白质组。简言之，将样品标记有3花青（CY 2，Cy3和Cy5）的发光在三个不同的波长（蓝色，绿色和红色）。含有N-羟基琥珀酰亚胺酯基团，这些氟最小的染料发生反应以产生共价酰胺键4蛋白的赖氨酸残基的ε-氨基。赖氨酸残基被标记之间只有1-3％，从而防止多个标签除了每蛋白质和主要净电荷修改5，6。 Cy3和Cy5经常使用的Cy2，而标签的样本来比较相同比例的组合来标记两个独立的样本。两个主要的优点是，所有标记的​​样品混合并IEF和SDS-PAGE均在1凝胶在一次为每个步骤，避免了相互之间的变异性是由于实验来胶凝比较。此外，它提出的大约1毫微微摩尔蛋白7的高检测阈值。凝胶进行扫描和2D软件比较2D凝胶的荧光图像，其中的Cy2作为内标，允许鉴定的斑点为他们的后路鉴定通过质谱法之间的统计学差异。使用内部标准的2D分析软件实现了快速检测的超过95％的统计置信8的采样之间的差值小于10％。 由微型二维超声心动图的定性分析是蛋白质鉴定的关键一步。其原理是如前对等电点和分子量分离所描述的相同，但在这种情况下，蛋白质是从一个小的聚丙烯酰胺凝胶转移到膜和免疫印迹之后被执行。而一维凝胶电泳提供改变蛋白质表达为一个或多个表位的蛋白质在功能上的抗体，该资料N迷你 - 二维超声心动图赋予了两个额外的参数。首先，蛋白质isovariants在pI值的函数改变，表明翻译后修饰可能发生。其次，质谱鉴定可能指示合理的酶原和蛋白质的分解代谢产物。因此，通过2D-DIGE观察到的修改很可能表明相关变化的全球蛋白质组配置文件的机制。几个样品为同一蛋白表位/秒由微型双向电泳免疫印迹之间的走线可能反映酸度变化脱落光成翻译后的变化观察到轻微的，甚至不是由单维免疫印迹9,10。此外，这种分析通知关于由于代谢残基11,12的pI和分子量的知识的潜在切割位点。 这两种技术的结合，提供了一个互补的蛋白质组学分析。一方面2D-DIGE提供了一次典型电子商务的差异使多肽的表达是不同的精确分离。这些差别主要由成斑或给定的一个用荧光凝胶的软件分析的强度的增加/减少的出现或消失。然而，这些观察结果本身不太可能解释所观察到的变形的性质。由于这些原因，一旦该多肽分离和质谱鉴定，采用微型2DE使得能够精确地确认1）中分离的蛋白的同一性和差异的2）的性质：变化中的isovariant /亚型水平表达，翻译后修饰和裂解过程为实例。然而，有必要开发一种开始裂解缓冲液都用2D-DIGE和与微型2DE兼容，以便限制从使用提取的协议是非常不相似而产生的潜在的分散体。 在本文章中，我们取消刻划一个适于协议用于2D-DIGE和微型2DE技术脑蛋白从人类和小鼠组织中来制备，萃取和性能。

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consensus brain-derived protein, extraction protocol for the study of human and murine brain proteome using both 2d-dige and mini 2de immunoblotting

二维凝胶电泳（2DE）是一个功能强大的工具来揭示蛋白质组的修改可能涉及到不同的生理或病理条件。基本上，这种技术是基于根据其等电点在第一步骤中，根据它们的分子量经SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）的蛋白质的分离，其次。在这份报告中对人类的验尸和小鼠脑组织中少量的优化样品制备协议描述。此方法使得能够同时执行二维荧光差异凝胶电泳（2D-DIGE）和迷你2DE免疫印迹。这些方法的组合，使人们不仅发现新的蛋白质和/或蛋白质修饰在它们的表达由于其与质谱检测的兼容性，又是一个新的洞察标记验证。因此，微型2DE耦合到西方墨点法证识别和验证后翻译后修饰，蛋白质分解代谢，并提供在不同的条件和/或治疗方法的定性比较。本文中，我们提供了研究中的AD和路易体痴呆，如β-淀粉样肽和α-突触核蛋白中找到的蛋白质聚集体的成分的方法。我们的方法，因此可以适用于蛋白质组的分析和不溶性蛋白质从人脑组织和小鼠模型中提取了。同时，它可提供用于所涉及的神经变性疾病以及潜在的新型生物标志物和治疗靶标分子和细胞途径的研究的有用信息。

对脑组织蛋白质组学仍然具有挑战性，因为没有理想的缓冲区存在收回100％的蛋白质，特别是膜相关或细胞骨架蛋白。在第一组实验都集中于寻找与这两种方法和使以回收蛋白质的大面板兼容合适的裂解缓冲液。因此，3裂解缓冲液进行测定，以确定最合适的一个。首先，它是用来将共同的生化和分子生物学缓冲的Tris-HCl 20在10mM的含1％（重量/体积）SDS（图1A）。此外，Tris-盐?...

Watch this Scientific Journal Video about Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting at JoVE.c

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

用尿素/硫脲/ SDS缓冲液为人类和小鼠脑组织中一种常见的蛋白提取协议允许由蛋白质2D-DIGE并随后将其表征迷你2DE免疫印迹鉴定。这种方法使一个人从活组织切片检查和实验模型获得更多的可重复性和可靠的结果。

共识脑源性蛋白，人类和小鼠脑蛋白质组使用两个2D-DIGE和Mini双向电泳免疫印迹法研究提取协议

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

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Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

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Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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