Name: pull-down of calmodulin-binding proteins
Uploaded: 2012-01-23T13:00:00
Description: Watch this Scientific Journal Video about Pull-down of Calmodulin-binding Proteins at JoVE.com

作者想感谢她的帮助下，在优化这个协议蒂芙尼樱桃。这项工作是由国家衰老研究所（AG032320）以及推进健康威斯康星州。

一个过程的开始与匀浆的基本原理，请参考图1。估计，从细胞中提取的准备钙调素结合蛋白洗脱的时间是6至7小时。 1。组织编制<ol><li>注入病毒包含利益的重组蛋白（在这个例子中，绿色荧光蛋白（GFP）标记伍），并允许组织一夜之间表达蛋白表达质粒器官海马。 </li><li>病毒注射后约12至18小时（取决于病毒表达时间），准备收集组织。加入1ml清扫缓冲区（10MM葡萄糖，4mm?...

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	<li>Vincenzi, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+in+the+regulation+of+intracellular+calcium.">Calmodulin in the regulation of intracellular calcium.</a> Proc. West Pharmacol Soc. 22, 289-294 (1979).</li><li>Cheung, W. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+myosin+light+chain+kinase+requires+the+calcium-binding+protein+calmodulin+for+activity.">Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity.</a> Proc. Natl. Acad. Sci. U.S.A. 76, 1653-1657 (1979).</li><li>Fukunaga, K., Yamamoto, H., Matsui, K., Higashi, K., Miyamoto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+a+Ca2+-+and+calmodulin-dependent+protein+kinase+from+rat+brain.">Purification and characterization of a Ca2+- and calmodulin-dependent protein kinase from rat brain.</a> J. Neurochem. 39, 1607-1617 (1982).</li><li>Yang, S. D., Tallant, E. A., Cheung, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcineurin+is+a+calmodulin-dependent+protein+phosphatase.">Calcineurin is a calmodulin-dependent protein phosphatase.</a> Biochem. Biophys. Res. Commun. 106, 1419-1425 (1982).</li><li>Huestis, W. H., Nelson, M. J., Ferrell, J. E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin-dependent+spectrin+kinase+activity+in+human+erythrocytes.">Calmodulin-dependent spectrin kinase activity in human erythrocytes.</a> Prog. Clin. Biol. Res. 56, 137-155 (1981).</li><li>Yamniuk, A. P., Vogel, H. 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提供的协议利用CaM的琼脂糖珠调查的Ca 2 +依赖CaM结合蛋白。许多蛋白质结合CAM中的Ca 2 +依赖性。这些相互作​​用CaM结合蛋白的数量和他们的许多信号通路中的关键作用给予高度重视。在这个协议中，CAM - Sepharose树脂用于组织匀浆中的Ca 2 +的存在或缺乏独立于CaM结合蛋白。这个简单的方法的结果将进一步了解蛋白质如何与CAM中的Ca 2 +依赖的方式进行交互。这种方?...

<table border="1"><tr><td> 产品 </td><td> 公司 </td><td> 目录编号 </td><td> 注释 </td></tr><tr><td>钙调蛋白的琼脂糖珠</td><td> GE医疗集团</td><td> 17-0529-01 </td><td></td></tr><tr><td>反CamKII阿尔法</td><td> Sigma - Aldrich公司</td><td> C6974 </td><td></td></tr><tr><td>抗- NG含量</td><td> Millipore公司</td><td> 07-425 </td><td></td></tr><tr><td>凝胶载入中吸取技巧</td><td>费舍尔</td><td> 02-707-138 </td><td>使用上清愿望</td></tr><tr><td>离心管（2.0毫升） </td><td>费舍尔</td><td> 05-408-146 </td><td>使用所有涉及钙调蛋白的琼脂糖珠的步骤</td></tr></table>

pull-down of calmodulin-binding proteins

钙（ 钙离子）离子通过多种机制调节细胞功能的重要。大部分的Ca 2 +信号是通过介导的钙结合蛋白称为钙调蛋白（CAM） 1,2。 CAM是涉及多层次的，在几乎所有的细胞过程，包括细胞凋亡，代谢，平滑肌收缩，突触可塑性，神经生长，炎症和免疫反应。的蛋白质，帮助调节与CAM的互动关系，通过这些途径。许多这些相互作用依赖于CaM的构象，这是明显不同的CA 2 +（的Ca 2 + - CAM）作为反对它的Ca 2 +自由状态（ApoCaM）3时，势必。 虽然大多数靶蛋白结合的Ca 2 + - CAM，某些蛋白质只能绑定到ApoCaM。有的通过他们的智商域绑定CAM，包括neuromodulin的NG（NG）5，4，和一定的肌球蛋白<sup&gt; 6。这些蛋白质已被证实，分别在突触前功能性突触后功能8，和肌肉的收缩9发挥的重要作用。他们的结合能力，并释放钙的缺乏或存在的 CAM +是在其功能的关键。相比之下，许多蛋白质结合的Ca 2 + - CAM，并要求其激活这个约束力。例子包括肌球蛋白轻链激酶10的Ca 2 + / CaM依赖的蛋白激酶（CaMKs）11和磷酸酶（如钙调磷酸酶）12，和血影蛋白激酶13，其中有各种直接和下游的影响14。 这些蛋白质对细胞功能的影响往往依赖于自己的能力结合到CAM中的Ca 2 +依赖性。例如，我们测试的NG - CAM约束力的突触功能的相关性和不同的基因突变如何影响这个约束力。我们生成一个GFP标记的吴CON结构与特定的突变，在IQ域会改变伍CAM中的Ca 2 +依赖性的结合能力。这些不同的基因突变的研究给了我们很大的启示，为参与突触功能的8,15的重要进程。然而，在这些研究中，它是必不可少的证明突变的蛋白质有约束力凸轮预期改变。 在这里，我们目前为检测蛋白质的能力绑定到CAM的Ca 2 +的存在或缺乏，作为例子使用CaMKII和NG方法。这种方法是一种亲和层析的形式简称为CAM的下拉检测。它使用的CaM -琼脂糖珠测试蛋白质结合CAM与此绑定的Ca 2 +的影响。这是相当多的时间效率和需要较少的蛋白质相柱层析及其他实验。总之，这提供了一个宝贵的工具，以探索的Ca 2 + / CaM的信号和蛋白质，在teract与CAM。

Watch this Scientific Journal Video about Pull-down of Calmodulin-binding Proteins at JoVE.com

Pull-down of Calmodulin-binding Proteins

钙调蛋白（CAM）下拉实验调查与各种蛋白质的CaM的相互作用的有效途径。此方法使用效率和具体分析CaM结合蛋白钙调素琼脂糖珠。这提供了一个重要的工具，探索细胞功能的CAM信号。

钙调蛋白结合蛋白的下拉

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Research

JoVE Journal

Biology

Electrophoretic Separation of Proteins

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

科研

生物学

Staining Proteins in Gels

在凝胶染色蛋白质

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Microcontact Printing of Proteins for Cell Biology

微接触印刷的蛋白细胞生物学

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Crystallization of Membrane Proteins in Lipidic Mesophases

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Rapid Generation of Amyloid from Native Proteins In vitro

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

内源性的免疫荧光分析及外源着丝粒着丝粒蛋白

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...