作者感谢医药生物技术和分子生物学的部门和工作人员，特别是莫妮克德Pelsmacker编制放射性标记的病毒。我们感谢埃伦MERCKX和Hadewych Halewyck有趣的发言和讨论，并以他在实验室的帮助下格里特德Bleeser。这项工作是由布鲁塞尔自由大学（OZR1807）的全宗，voor Wetenschappelijk Onderzoe​​k Vlaanderen（G.0168.10N）和世界卫生组织（TSA的200410791）OZR批财政支持。莉莎好德是博士前的全宗，voor Wetenschappelijk Onderzoe​​k Vlaanderen（FWO）研究员。

图1描述的原则（A）和方法的概述（二）。 1。缓冲区的制备<ol><li>准备结合/洗涤液溶解磷酸二氢钠（50毫米）和氯化钠于水（300毫米）和pH值调整到8.0。此外添加吐温20（终浓度为0.01％（M / V）），蛋氨酸（终浓度为2％（M / V））和白蛋白（终浓度为0.1％（M / V）），并调整到所需的体积。 </li></ol> 2。磁珠的制备按照说明书准备磁珠。简述如下： <ol><li>重悬磁珠使用一个旋涡。 </li><li>转移，对每个样品，10μL重悬磁珠成筒（40毫克/毫升）。 </li><li>使用磁铁收集磁珠，直到上清液是明确的（+ / - 30秒），去除上清仔细。 </li><li>洗磁珠两次：悬浮在150μL结合/洗涤液珠。磁珠迁移到使用磁铁，直到上清液是明确管的一侧，小心取出上清液。 </li><li>用40μL结合/洗涤液，每个样品，悬浮珠（10毫克/毫升）。 </li></ol> 3。亲和捕获法注：为了测量（宇宙）的背景放射性（0％放射性），无放射性的病毒被添加到控制样品1。相反，相同体积的结合/洗涤液补充。 注：100％的放射性控制样品2除nanobody添加所有组件的定义。相反，相同体积的结合/洗涤液补充。 <ol><li>使2000年35 S的标记（β辐射）脊髓灰质炎病毒萨宾株类型1 9日至80μL结合/洗涤液稀释成96孔板CPM。 </li><li>新增10＆MU;升井nanobody稀释。 </li><li>混合使用振动筛约10秒。 </li><li>允许的样品在室温下1小时孵化。 </li><li>加入40μl和水洗悬浮磁珠孵育10分钟，在室温下不断晃动。 </li><li>分开珠和使用磁铁的上清转移到管清除上清。 </li></ol> 4。放射性测量<ol><li>转移50μL上清步入一个计数瓶3.6。 </li><li>加入3毫升闪烁液和混合。测量放射性β-闪烁计数器。 </li></ol> 5。回收的珠子<ol><li>在500 XG 2分钟或直到得到一个明确的上清收集管和离心机使用的磁珠。 </li><li>除去上清，悬浮在6毫升0.5 M氢氧化钠珠。 </li><li>转移到含多处悬挂E管和超声波水浴孵育5分钟。 </li><li>使用磁铁收集磁珠，去除上清。 </li><li>每个试管中加入1毫升2％SDS溶液和悬浮使用一个旋涡。在5分钟烧开。 </li><li>收集珠子，去除上清。珠悬浮在1毫升0.2 M EDTA（pH值= 7）。 </li><li>在5分钟的超声波浴孵育管一次，去除上清。 </li><li>每管加入1毫升的水和悬浮的珠子。收集珠子，去除上清。重复此清洗步骤两次。 </li><li>去除上清，1毫升10毫米CoCl 2溶液中悬浮的珠子。振动筛，放10分钟。 </li><li>去除上清，用磁铁收集珠和悬浮在1毫升结合/洗涤缓冲。 </li><li>去除上清，洗珠两次使用1毫升20％（V / V）乙醇</li><li>悬浮在其原体积20％（V / V）等珠hanol获得再生珠，在浓度为40毫克/毫升。 </li></ol> 6。结果解读<ol><li>控制样品1，在没有放射性病毒，将用于为背景的放射性和纠正设置0％的辐射值。控制样品，其中不包含的nanobody和地方，因此所有放射性病毒仍然存在，在上清设置为100％的辐射值。 </li><li>放射性上清液中的比例（％）是衡量一个整体降水量（= 100 - ％）放射性病毒由nanobody的。 </li></ol> 7。代表结果代表亲和捕获检测的结果显示在图2，图3和4。在图2所示的PVSS38C，脊髓灰质炎nanobody的具体亲和力。亲和力PVSS38C测试三个不同的抗原poliovirus：母语抗原（N抗原），加热抗原（H抗原）和14S亚基。的N-抗原是完整的病毒是有传染性的。当病毒被加热（56℃20分钟），或附着在固体表面（未发表结果），衣壳变化的构象，造成损失的病毒RNA（部分）和衣壳蛋白VP4，空衣壳的形成，则称为H抗原。 14S亚基组装中间体。这些抗原被确认由不同的抗体免疫后10集，并具备因此，不同表位抗原位点。在上清液中发现的放射性物质的百分比数字表示作为浓度nanobody功能。由此可以看出，在样本含有放射性14S亚基上清放射性与该nanobody PVSS38C浓度的增加而降低。这可以被翻译为脊髓灰质炎病毒的14S亚基CON量增加连接到nanobody PVSS38C和间接的磁珠。可以捕捉滴度（= nanobody必要浓度捕捉放射性病毒的50％）为0.099 nm的图形计算。没有PVSS38C为N-抗原和脊髓灰质炎病毒的H抗原形式的亲和力观察。从这些数据中，它被解释，PVSS38C完全是存在的14S亚基和两个脊髓灰质炎病毒抗原形式与抗原相互作用。 为了证明上清液的放射性损失只是由于具体实现其抗原nanobody亲和力，同样的检测与NB1，对硫化sulfataricus转录调控lrpB产生称为nanobody的，没有互动任何脊髓灰质炎病毒抗原。这个实验的结果如图3所示。所有放射性病毒仍然有没有反应，显示上清NB1对三种脊髓灰质炎病毒抗原形式。 共八次在不同天为一个给定的nanobody（即PVSP29F）的重复实验，并通过不同的人进行了测试结果的重复性。 如图4所示的结果。上清液中的放射性百分比平均值计算每个nanobody浓度，并在其标准差的信件表示。 <img alt="图1。" src="/files/ftp_upload/3937/3937fig1.jpg" /> 图1。原则（一）概述和方法（二）检测 。答：他的标签，明确承认将能够互动与钴磁珠，将沉淀的放射性标记的病毒有一定的脊髓灰质炎病毒抗原的纳米抗体。 B.他的标记的纳米抗体被添加到放射性标记的脊髓灰质炎病毒和培养。钴磁珠添加和绑定/绑定抗原进行磁选。上清液的放射性测量和抗原捕获量可得。 <img alt="图2" src="/files/ftp_upload/3937/3937fig2.jpg" /> 图2。不同的脊髓灰质炎病毒抗原磁珠亲和捕获由nanobody PVSS38C。上清液中发现的放射性物质的百分比表示作为浓度PVSS38C功能。为14S浓度响应关系可以发现，14S目前与抗原完全PVSS38C互动。没有显着的交互作用的N-，也可以检测到的H-抗原，虽然在很高的浓度可以忽略不计的非特异性捕捉注意到。 <img alt="图3" src="/files/ftp_upload/3937/3937fig3.jpg" /> 图3。不同的脊髓灰质炎病毒抗原的亲和力磁珠捕获nanobody NB1 </stronG&gt;。作为一个功能的NB1浓度的上清液中发现的放射性物质的百分比表示。 NB1被称为没有任何脊髓灰质炎病毒抗原或亚基相互作用。由于100％的放射性上清液中发现，没有病毒非特异性结合到NB1。 图2中的放射性的损失，因此只是由于具体实现其抗原nanobody亲和力。 <img alt="图4。" src="/files/ftp_upload/3937/3937fig4.jpg" /> 图4。重复性检测 。代表平均值的百分比在上清放射性作为浓度nanobody功能。实验在不同的日子，不同的人重复八次。竖线表示标准偏差。

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我们什么都没有透露。

在协议的相互作用抗原与nanobody的放射性被定义为放射性病毒上清损失。因此沉淀放射性（= 100 - ％）金额（绑定向磁珠）可以以间接的方式，由上清（％）放射性估计。另一方面，它也有可能在500 mM咪唑洗脱从磁珠免疫复合物的直接的方式来测量放射性沉淀的抗原结合部分。它以前被证明是有放射性物质的总量和上清和沉淀分数发现放射性的总和之间的完美匹配。因此，它是可以接受的和节省?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;试剂名称</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;<td<strong&gt;评论</strong</td&gt;</tr&gt;<tr&gt;<td&gt;磁珠分离His标签和下拉</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; 101.03D</td&gt;<td&gt;磁珠</td&gt;</tr&gt;<tr&gt;<td&gt; Optiphase“HiSafe 2</td&gt;<td&gt; Perkin Elmer公司</td&gt;<td&gt; 1200 - 436</td&gt;<td&gt;闪烁液</td&gt;</tr&gt;</tbody&gt;</table

a liquid phase affinity capture assay using magnetic beads to study protein-protein interaction: the poliovirus-nanobody example

在这篇文章中，提出了一个简单，定量，液相亲和力捕获检测。提供一种蛋白质，可以用来标记和标记的另一种蛋白质，这种方法可以实现蛋白质相互作用的调查。它是基于一个钴磁珠标记蛋白的识别和标签蛋白和标记的第二个特定蛋白质之间的相互作用，另一方面手。首先，标记和标签的蛋白质混合，在室温下孵育。磁珠，识别标签，添加和标记蛋白结合部分分开绑定使用磁铁的分数。被捕获的标记蛋白量可确定以间接的方式，通过测量的标记蛋白的信号仍然在绑定分数。描述液相亲和力检测是非常有用的，当敏感蛋白的构象转化的屁股ayed。检测的发展和应用，证明认识到纳米抗体1的脊髓灰质炎病毒和脊髓灰质炎病毒之间的相互作用。由于脊髓灰质炎病毒是敏感的构象转化2时，附着在固体表面（未发表结果），ELISA法的使用是有限的，因此，应优先考虑液相系统。液相基于polioresearch 3,4经常使用的系统的一个例子是一个免疫试验5微蛋白。即使这个测试已经证明了它的适用性，它需要一个FC结构，这是在纳米抗体6,7缺席。然而，作为另一个机会，这些有趣的和稳定的单域抗体可以很容易地设计不同的标签。广泛使用（他）6标记显示为二价离子，如镍，钴，可以在轮到他们可以轻松地在磁珠表面的亲和力。因此，我们开发了这个简单的定量基于钴磁珠亲和捕获检测。脊髓灰质炎病毒35 S的标记，使入无人之境的互动与纳米抗体，并做出了定量的检测可行的。方法是易于执行，并可以以较低的成本，这是有效再生磁珠的可能性进一步支持建立。

Watch this Scientific Journal Video about A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example at JoVE.com

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

在这篇文章中，提出了一个简单，定量，液相亲和力捕获检测。这是一个可靠的技术基础上，一方面与亲和力之间的标记蛋白，对其他的第二个标记蛋白（如脊髓灰质炎病毒）之间的磁珠和标签蛋白（如纳米抗体）的相互作用。

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 ...

a-liquid-phase-affinity-capture-assay-using-magnetic-beads-to-study

Research

JoVE Journal

Biology

11.8K 次观看.  Vrije Universiteit Brussel. 在这篇文章中，提出了一个简单，定量，液相亲和力捕获检测。这是一个可靠的技术基础上，一方面与亲和力之间的标记蛋白，对其他的第二个标记蛋白（如脊髓灰质炎病毒）之间的磁珠和标签蛋白（如纳米抗体）的相互作用。

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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 ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

凝胶电泳迁移分析（EMSA）为RNA，蛋白质相互作用的研究：IRE / IRP示例

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

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 ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.