Name: protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro
Uploaded: 2011-08-14T19:00:00
Description: Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

在我们的实验室的工作是从国立卫生研究院，美国农业部国家粮食和农业研究所，国家科学基金会，巴德，能源部和BSF拨款到VC支持

1。表达和提取的蛋白质<ol><li>差异标记要为他们的检测测试的蛋白质。标签膜（ProIM）被固定在一个尺寸较大的标签（例如，消费税），非融合标签可以使用一个固定的阴性对照组（ProIMnc）的蛋白质。标签用于大型或一个小标签作为一种可溶性的探头（ProSOL）的蛋白质。保险丝相同的标记，以一个合适的阴性对照的可溶性蛋白（ProSOLnc），包括在互动中检测，以确认检测约束力的特异性。 </li><l...

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这种方法适用于检测蛋白质相互作用的蛋白质之间的组合，当至少有一个，其中的蛋白质很容易结合缓冲液中的可溶性，并成功地应用到其他结合蛋白17,18。不能测试都是在这些条件下的不溶性蛋白质之间的iInteractions本议定书。 此外，ProIM成功复性检测的关键。漂洗后的electrotransfer在TBS的膜是关键的一步，因为残留的SDS损害变性/复性过程。 最后?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>蛋白检测试剂盒</td><td> Bio - Rad公司</td><td> 500-0001 </td></tr><tr><td>蛋白酶抑制剂的鸡尾酒</td><td>西格玛</td><td> S8820 </td></tr><tr><td>迷你PROTEAN系统</td><td> BIO - RAD </td><td> 165-8000 </td></tr><tr><td>半干免疫印迹SD electrotransfer系统</td><td> BIO - RAD </td><td> 170-3940 </td></tr><tr><td>抗兔IgG抗体与辣根过氧化物酶的共轭</td><td>金斯瑞</td><td> A00098 </td></tr><tr><td>抗GST多克隆抗体</td><td>金斯瑞</td><td> A00097 </td></tr><tr><td>抗- strepII </td><td>金斯瑞</td><td> A00626 </td></tr><tr><td> BioTrace，NT硝化棉转印膜</td><td>帕尔科学</td><td> 27377-000 </td></tr><tr><td>的Immobilon西部发光辣根过氧化物酶底物</td><td> Millipore公司</td><td> WBKL S0 050 </td></tr></tbody></table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

验证不同的蛋白质之间的相互作用是至关重要的，为在分子水平上的生物学功能的调查。有几种方法， 在体外和体内 ，以评估蛋白结合，并应进行至少有两种方法，相互补充的缺点，以获得可靠的见解。 对于一个在体内实验中，双分子荧光互补（附设）检测最流行 ​​的，至少侵入的方法，使检测活细胞内蛋白质相互作用，以及识别细胞内相互作用的蛋白质1,2的本地化。在这个实验中，测试蛋白质融合GFP或它的变种N -和C -末端非荧光半，两个融合蛋白汇聚由于测试的蛋白质“相互作用时，荧光信号是重组 3-6 。因为它的信号很容易epifluorescence或共聚焦显微镜检测，附设已经成为一个强大的工具，在活细胞3中蛋白质相互作用的研究细胞生物学家之间的选择。然而，这个实验中，有时会产生假阳性结果。例如，荧光信号可以安排尽可能远离对方因7纳米包装在一个小的亚细胞车厢关闭两个GFP片段重组，而由于特定的相互作用 7 。 由于这些限制，从活细胞成像技术所取得的成果，应确认由一个独立的方法的基础上，检测蛋白质相互作用的原理不同。免疫共（联合IP）或谷胱甘肽转移酶（GST）的代表等，通常用于分析蛋白质相互作用，在体外Pull - down实验的替代方法。然而，IIN这些实验，然而，测试的蛋白质一定要容易溶于supportsused结合反应的缓冲区。因此，具体涉及一种不溶性蛋白质的相互作用，不能由这些技术进行评估。 在这里，我们说明了蛋白质膜覆盖结合试验，从而绕过这个困难的协议。在这种技术中，可以可靠地测试可溶性和不溶性蛋白质之间的相互作用，因为固定在膜基质的蛋白质之一。这种方法， 在体内实验，如附设结合，提供了一个可靠的方法，调查和定性可溶性和不溶性蛋白质之间的相互作用忠实。在这篇文章中，烟草花叶病毒（TMV）之间具有约束力的运动蛋白（MP），发挥多种功能，在病毒的细胞与细胞之间的运输8-14，以及最近发现植物细胞interactor，烟草含有锚蛋白重复蛋白（ANK 15），证明使用这种技术。

Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

测试是必不可少的蛋白质 - 蛋白质相互作用蛋白质功能的夹层。在这里，我们引入<em&gt;在体外</em&gt;蛋白质 - 蛋白质结合检测探头与可溶性蛋白的膜固定蛋白质。这个实验提供了一个可靠的方法来测试不溶性蛋白质在溶液中的蛋白质之间的相互作用。

蛋白膜覆盖含量：一个协议测试可溶性和不溶性蛋白质之间的相互作用在体外</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Research

JoVE Journal

Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

In vitro Methylation Assay to Study Protein Arginine Methylation

In vitro Methylation Assay to Study Protein Arginine Methylation

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

荧光互补成像检测在烟草烟叶中瞬时测定蛋白质-蛋白质相互作用动力学

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...