我们要感谢共聚焦显微镜的帮助下，斯科特亨德森博士。这项研究是从美国国立卫生1RO1CA127501研究院的资助，以WAB的目标，以及来自梅西癌症中心和医学院（VCU），WAB的显微镜，在VCU - DEPT试点项目的资金支持。神经生物学及显微解剖设施，支持，从NIHNINDS中心核心授予5P30NS047463资金中的一部分，。

该协议包括三个主要步骤。第一，一步一个哺乳动物表达载体的氨基端的CFP资格和/或YFP的单体版本将只简要地讨论你感兴趣的基因的克隆。第二和第三个步骤;载体DNA转成EA.hy926内皮细胞，并与FRET的共焦成像，在下文作更深入的概述。 1。 CFP和YFP嵌合受体的建设感兴趣的受体被克隆氨基酸末端CFP和YFP单体的增强版本。 FRET的测定是经验性的，是严重依赖后，连接器之间的长度的跨膜跨越区域，并开始荧光 1 。因此，几个不同长度的连接器，必须探索新的受体对每个正在调查。 <ol><li>进入的pcDNA3.1吸湿R或含有单体C或YFP的（这可以从我们的实验室获得的矢量） 新 ř载体，NheI / EcoRI位点克隆到您感兴趣的受体。 *注意:如果有必要，NheI包括SpeI和XbaI其他兼容的几个酶。 </li><li>连接反应转换成适当的endA1 recA基因主管细胞系（我们经常使用MACH1）。其次DNA测序验证突变的情况下插入存在的屏幕殖民地。 </li><li>取得成功的分子克隆构象后，准备在无菌水或使用Qiagen公司，或类似，DNA的试剂盒TE转染级的载体DNA。评估浓度和纯度，采用紫外光谱。典型的DNA产量应在100-200微克，这是常规稀释到1μg/μL的工作股票的范围。 </li></ol> 2。 EA.hy 926细胞的转染<ol><li>拆分成MatTek盖玻片（0号）2毫升培养基的35 mm培养皿EA.hy 926细胞，生长在完全DMEM培养液（DMEM培养液10％小牛血清+ Pen. /链球菌。） 。保守，你可以得到融合菜从15厘米〜15道菜（〜90％融合翌日）。准备一盘，以评估每一个人的受体的表达，以及一盘每个受体对。 </li><li>允许细胞在5％的CO 2气氛孵育过夜在37 ° C 。翌日，文化应约70-90％汇合。 </li><li>转天，准备nucleolipid络合物8μL的Fugene6预热OptiMEM 200μL混合嵌合受体的载体DNA（2μL），2微克。对于受体对，染（见下面的讨论），每个受体共2微克，1微克。 </li><li>轻轻颠倒混合。 </li><li>继续在室温下孵育30分钟。 </li><li>在发病潜伏期间，准备转染的细胞。吸新鲜的媒体和每盘用温PBS冲洗，并简要地前增加2毫升不含抗生素的预热完全DMEM培养液（DMEM培养基+胎牛血清）。重要的是避免青霉素和链霉素的存在，因为它们与Fugene6治疗期间，相当多的细胞毒性。 </li><li>添加复杂的细胞明智的下降和一个额外的20-24小时内孵化，在37 °彗星在 5％的CO 2的气氛。 </li><li> 24小时后，小心吸媒体从细胞和吸管用抗生素新鲜完整的DMEM培养液。转染后，细胞存活率应保持比较高的，即应观察几乎无漂浮细胞。 </li><li>基因表达是一个额外的转染后48-60小时后通常最大。然而，成像通常是可行的，在短短24小时。应为最佳的基因表达的时间当然实验。 </li></ol> 3。共聚焦成像，FRET技术分析活细胞成像的Leica TCS SP2 AOBS共聚焦激光扫描配备蓝色二极管（405纳米），氩气（458，476，488，514纳米）显微镜。氦氖绿（543纳米），橙色氦氖（594纳米），和红色的氦氖（633 nm）的激光器，一个HCX PI阿婆63x/1.3 NA甘油浸入式物镜，机动XY阶段（Märzhäuser），和环境控制（温度，湿度和CO 2）的阶段，孵化器（PeCon）。实验控制是至关重要的，以消除荧光基团之间的排放渠道，以及评估的表达问题和非特异性荧光基团的多聚串扰。因此，单个荧光团的转染是利用设立每一个形象会话。负的FR​​ET控制（使用已知的无相互作用的受体）将需要被执行，以确定背景的FRET的发生是由于过度表达。 <ol><li>为了消除CFP和YFP的排放渠道之间的串扰，将进入阶段孵化器的CFP的表达调控。使用白色的光，调整的Z板将重点放在细胞。 </li><li> SP设置窗口设置CFP排放检测到465-505 nm和YFP的525-600纳米。 </li><li>同时监控的排放渠道，激发CFP的458纳米。 AOTF的使用，设置激光功率超过CFP YFP排放通道，以消除明显出血。保存该设置。 </li><li>通过调整电源的激发下，在514 nm处表示只YFP的细胞执行在CFP通道YFP的串扰（使用YFP的表达调控）相同的控制。保存该设置。 </li><li>未来的地方细胞共表达CFP和YFP进入阶段孵化器。使用徕卡共聚焦软件的序列设置，找到适当的膜定位表达CFP和YFP的水平相似。 </li><li>使用徕卡软件受体光漂白程序，设置的供体和受体设置保存CFP和YFP设置，可敬。 </li><li>放大到细胞，突出一个YFP的照片毁灭是发生在细胞膜，并开始计划的ROI（感兴趣区域）。 </li><li>承兑人的照片销毁，调整的AOTF在514纳米至100％，并允许继续下去，直到YFP的排放量的70％已所剩无几的激光漂白。为了加快漂白，投资回报率通常选择在光栅的激光轴（X轴）。 </li><li>在光漂白过程中，监测细胞的运动，并拒绝在XY平面明显运动中获得的测量，这些测量报告虚假的FRET效率。 </li><li>前和后的漂白图像记录捐助国（CFP）和受体（YFP），FRET的效率计算公式为:FRET的效率 =（D - D 后前 ）/ D转换后 ，所有D后&gt; ð前， 其中 D前和D 后是漂白后分别捐助力度。 </li><li> JMP软件（SAS）是用来确定实验变异程度。 </li></ol> 4。代表性的成果转染效率通常在30至40％之间。尽管以前的研究结果被别人，我们还没有看到一个载体转染和表达，有利于其他。事实上，我们经常观察一个嵌合体荧光基团或其他专属的表达。典型的FRET受体系统的效率之间的不同。对于领带的受体系统，典型值是20-28％和19-23％的内皮细胞上皮细胞。对于阴性对照，典型的效率都低于2-3％。有经验的变异程度将大大降低。 <img src="/files/ftp_upload/2149/2149fig1.jpg" alt="图1" /> 图1。转EA.hy 926细胞的代表图像 。）EA.hy 926细胞单层DIC的形象。二）（一）显示细胞的荧光图像。 c）覆盖（A）和（二）展示〜20％至30％的转染效率。 <img src="/files/ftp_upload/2149/2149fig2.jpg" alt="图2" /> 图2。代表承兑人的照片漂白FRET CFP和YFP发生在EA.hy926细胞的分析。CFP排放通道（顶部面板）和YFP的发射通道（上下两板）分别进行了监测前，后受体漂白。漂白实验受到限制，和FRET技术的绿色方框内的区域，计算值。 FRET的效率是一个绝对的范围从高（红色- 1.0）低（紫色- 0.0）上一个CFP / YFP的放大叠加显示，合并方向的目的形象。

<ol>
	<li>Kim, M., Carman, C. V., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+transmembrane+signaling+by+cytoplasmic+domain+separation+in+integrins.">Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.</a> Science. 301, 1720-1725 (2003).</li><li>Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+lipidmodified+monomeric+GFPs+into+membrane+microdomains+of+live+cells.">Partitioning of lipidmodified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296, 913-916 (2002).</li><li>Seegar, T. C. M., Eller, B., Tzvetkova-Robev, D., Kolev, M., Henderson, S. C., Nikolov, D. B., Barton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie1-Tie2+interactions+mediate+functional+differences+between+angiopoietin+ligands.">Tie1-Tie2 interactions mediate functional differences between angiopoietin ligands.</a> Molecular Cell. 37 (5), 643-655 (2010).</li><li>Edgell, C. J., McDonald, C. C., Graham, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+cell+line+expressing+human+factor+VIII-related+antigen+established+by+hybridization.">Permanent cell line expressing human factor VIII-related antigen established by hybridization.</a> Proc Natl Acad Sci U S A. 80 (12), 3734-3737 (1983).</li></ol>

有几个步骤是成功的关键。其中最突出的是两者之间的嵌合受体表达的相对水平。为了规避这个问题，可能使稳定的细胞系，表达利益蛋白，或确定载体DNA的最佳比例，允许相当于表达。同样，由于非均匀转跨菜，蛋白水平，很少会相当于细胞间。因此，一定要注意，"低"，以区别于"高"expressors。 ，显示"平均"的蛋白质含量通常会产生可靠和可重复性的FRET效率。我们的实验室一直奉行以减轻这个问题的方法之一是使用腺和lenti病毒"甚至是"基因的表达。此外，一个替代的方法来确定受体照片漂白FRET效率是致敏的排放。虽然，尽管敏排放实时监测单细胞的潜力，我们已经发现受体光漂白更敏感和更可靠。 最后，利用FRET技术来监测蛋白相互作用，可以是具有挑战性的的，需要谨慎选择的膜结合受体的链接长度。此外，除了C / YFP往往大大影响内质网和高尔基体蛋白表达水平的聚集和结果。然而，短暂的，不稳定的，互动的，FRET是理想的方法利用。

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11960-069</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15070-063</td>
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<td>&nbsp;</td>
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<td>N/A</td>
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<td>&nbsp;</td>
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imaging protein-protein interactions in vivo

蛋白质 - 蛋白质相互作用，是所有重要的细胞过程的一个标志。然而，许多这些互动是短暂的，或大力弱，防止他们的身份，并通过分析传统的生物化学方法，如免疫共沉淀。在这方面，已经彻底改变了基因encodable的荧光蛋白（GFP，RFP等）及其相关荧光光谱重叠，我们有能力监测在体内的弱相互作用，使用福斯特共振能量转移 （ FRET）1-3。在这里，我们详细介绍我们使用一个基于FRET的接近检测，监测上的内皮细胞表面的受体受体相互作用。

Watch this Scientific Journal Video about Imaging Protein-protein Interactions in vivo at JoVE.com

Imaging Protein-protein Interactions in vivo

本协议描述了如何形象蛋白质相互作用使用基于FRET的接近检测。

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

imaging-protein-protein-interactions-in-vivo

Research

JoVE Journal

Biology

Imaging Protein-protein Interactions in vivo

21.3K 次观看.  Virginia Commonwealth University. 本协议描述了如何形象蛋白质相互作用使用基于FRET的接近检测。

视频: Imaging Protein-protein Interactions in vivo

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

科研

生物学

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

的蛋白质 - 蛋白质相互作用抑制剂的发展，通过替换:应用程序的设计和开发非ATP竞争性CDK抑制剂

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

自噬研究中蛋白质-蛋白质相互作用的研究

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

成像蛋白质 - 蛋白质相互作用在体内

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