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><li>选择蛋白表达系统表达标记蛋白，即大肠杆菌大肠杆菌 ，杆状病毒等，根据潜在的翻译后修饰和蛋白质的产量的要求。 </li><li>从选择的有机体，使用SDS - PAGE电泳上样缓冲液（20％的甘油，4％SDS，20毫米的Tris - HCl，pH6.8）含有蛋白酶抑制剂的鸡尾酒（步骤1.2）中提取 ProIM和ProIM NC。在室温为15分钟的保留细胞悬液，加入0.5％2 - 巯基乙醇和煮沸5分钟。 </li><li>从选择的有机体，使用标准的协议16（步骤1.2）中提取ProSOL和ProSOL NC。这些蛋白质应该很容易在结合缓冲液中的可溶性（2毫米EDTA，140毫米，10毫米氯化钠，pH值7.4的Tris - HCl，2毫米的数码地面电视服务，1％BSA，0.1％吐温20）的步骤4.1中使用。 </li><li>确定重组的蛋白质，如Bio - Rad公司蛋白测定试剂盒，使用一种标准的方法的浓度。 Bio - Rad公司蛋白检测试剂盒等。如果原油中提取，纯化的蛋白质，而不是，是用来检测，估计相应频段的光密度扫描SDS -聚丙烯酰胺凝胶染色用考马斯亮蓝R - 250和使用已知浓度的蛋白质，这种提取物的兴趣BSA的浓度作为参考。 </li></ol> 2。固定在膜ProIM和ProIMnc <ol><li>解决两套提取，每个SDS -聚丙烯酰胺凝胶含有1微克ProIM和ProIM NC根据标准协议16。 </li><li>电泳后，转移地方凝胶在转移缓冲液100毫升（60毫米甘氨酸，10毫米三，0.0006％SDS，20％甲醇）中，轻轻摇动孵育在室温为20分钟。 </li><li> Electrotransfer包含在凝胶中根据标准协议 16至硝酸纤维素膜上的蛋白质。 </li></ol> 3。膜结合的蛋白质重新折叠<ol><li> electrotransfer后，孵育15分钟在15毫升的缓冲膜（30毫米的Tris - HCl pH7.4的0.05％吐温20），轻轻摇动，以去除残留的SDS。 </li><li>经过仔细排水，转移膜的变性缓冲（7米盐酸胍，2毫米EDTA，50毫米数码地面电视，50毫米的Tris - HCl pH值8.3）至25毫升。孵育2小时，在室温下轻轻摇动。 （注：在变性缓冲液孵育时，硝酸纤维素膜变得不透明）。孵育2小时，在室温下轻轻摇动。 </li><li>膜转移至25 ml的TBS（10毫米的Tris - HCl，150 mM氯化钠，pH值7.4），轻轻摇动孵育5分钟（注意：在此步骤中，膜恢复原有的白色）。 </li><li>膜转移至25毫升的结合缓冲液（见第1.2步），并在4℃，轻轻摇动孵育过夜另。 </li></ol> 4。通过ProSOL探测的ProIM <ol><li>切成条状，既包含ProIM和ProIM NC膜。 </li><li>稀释1-10微克或10毫升新鲜的结合缓冲液中，无论是ProSOL ProSOLnc准备的ProSOL和ProSOLnc杂交解决方案。膜转移到每个ProSOL或ProSOLnc杂交液和孵育1.5 h在室温轻轻摇动。 </li><li>取出膜杂交的解决方案，并在TBS电视台每15分钟冲洗三次。 </li></ol> 5。可视化免疫印迹蛋白质相互作用<ol><li>座膜在室温下1小时与2.5％的脱脂奶粉TBST（10毫米的Tris - HCl，140 mM氯化钠，0.05％吐温20，pH值7.4）。在制造商建议的浓度在0.5％的脱脂奶粉TBST稀释的主要抗体（抗- strepII多克隆兔抗体）。 </li><li>堵塞膜放置在抗体溶液，在室温下1小时或一夜之间孵育4℃，轻轻摇动。 </li><li>在15分钟20毫升TBST冲洗膜，并两次在室温下轻轻摇动5分钟。 </li><li>稀释与辣根过氧化物酶（HRP）的制造商建议的浓度在0.5％的脱脂奶粉TBST标记的第二抗体（抗兔IgG抗体）。将在二级抗体的膜解决方案，并在室温下轻轻摇动1小时孵化。 </li><li>在15分钟20毫升TBST冲洗膜，并两次在室温下轻轻摇动5分钟。最后的TBS冲洗后，可视使用HRP化学发光底物（例如，Millipore公司的Immobilon西部发光辣根过氧化物酶底物）的蛋白 - 蛋白相互作用。 </li><li>探头与融合ProIM，通过适当的辅助步骤5.1至5.4的抗体标记抗体相同的膜。可视化ProIM和ProIMnc中所述的步骤5.5来验证身份的蛋白膜覆盖法（步骤5.5）中获得的频段。在大多数情况下，剥离是没有必要，因为这一步得到的信号是比从步骤5.5中获得的剩余信号强多了。 </li></ol> 6。代表性的成果： 附设在烟草表皮细胞（图1A）ANK - MP的相互作用。由于MP是一种高度不溶性蛋白质，在细菌或植物中的表达时，蛋白膜覆盖法通过，以验证这种在体外的相互作用（图1B ）。蛋白提取物中含有1微克的GST - MP（ProIM）或者未熔化的商品及服务税（ProIM NC）的SDS -聚丙烯酰胺凝胶电泳，硝酸纤维素膜，由electrotransfer解决。当这些ProIMs探讨可溶性ANK strepII（ProSOL），GST - MP，但不是非融合商品及服务税，展出具有约束力（图1B，泳道1，2，比较泳道5，6）。此外，当同一组的ProIMs无关ProSOLnc，即标签strepII（NADH3 strepII） 拟南芥胞质 NADH激酶，没有约束力不遵守，探讨进一步证明ANK - MP相互作用的特异性（图1B，车道3，4）。 <img src="/files/ftp_upload/2961/2961fig1.jpg" alt="图1"/> 图1。烟草ANK特异性结合烟草花叶病毒在体内和体外的MP 。 （一）ANK - MP在生活附设检测烟草表皮细胞的相互作用。强YFP的信号重建时，MP和ANK，C端和N -端半YFP的，分别融合，在烟草表皮microbombardment其编码基因共表达。这附设信号在puncta积累在细胞外围，这是15胞间连丝的诊断。 （二）ANK - MP 在体外的相互作用检测蛋白膜覆盖检测。 15％SDS聚丙烯酰胺凝胶electrotransfer随后到硝酸纤维素膜，蛋白提取物中含有1微克的GST - MP（ProIM），或者未熔化的商品及服务税（ProIMnc）解决。 GST - MPProIM和GSTProIMnc孵育0.5微克/毫升ANK strepII（ProSOL），ANK约束力是通过探测与反strepII多克隆抗体的膜，抗兔IgG + M二级抗体偶联检测辣根过氧化物酶（泳道1和2）。无论是GST - MPProIM也不GSTProIMnc互动与融合strepII标签（ProSOLnc，泳道3和4），一个不相关的蛋白，拟南芥胞质NADH激酶。在这个实验中观察到的乐队的身份被确认通过探测与抗GST抗体（5和6车道）膜。变性缓冲处理，而不会被用缓冲液A洗涤，当膜的GST - MP绑定ANK丢失，而不明身份的GST - MP和消费税contining蛋白提取物中的蛋白质与ANK - strepII反应，表现出的重要性步骤3.1之前的变性过程（7和8）。

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没有利益冲突的声明。

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 ...

protein-membrane-overlay-assay-protocol-to-test-interaction-between

Research

JoVE Journal

Biology

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

22.0K 次观看.  State University of New York. 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，我们引入在体外蛋白质 - 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。

视频: 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

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

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

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

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

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

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

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

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

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

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

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 ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

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

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

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

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

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

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

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

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

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

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

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 ...

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

通过BiFC-FRET-FLIM测定MADS盒转录因子和钙传感器蛋白两种单体之间的三方相互作用

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

大肠杆菌重组蛋白填充囊泡的生产和成像

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium&#160;and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

作者聚焦：重组蛋白的生产 

The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.