这项工作是由马普学会的支持。

质粒1。创作<ol><li>亚克隆的cDNA为每个感兴趣的蛋白到两个pLuc和pYFP载体，用标准的分子生物学技术（ 图1）。有关详细方案见绿等[13]。 </li><li>序列中的所有构造，以验证所感兴趣的蛋白质在帧与吕克/ YFP序列中间没有终止密码子。 </li><li>如期望的，以确认该融合蛋白保留生物活性进行功能测定。在这里呈现的情况下YFP融合蛋白的亚细胞定位是确定通过荧光显微镜。 </li><li>通过设计相关的目标信号进入pLuc和pYFP表达载体使表达质粒适当吕克和YFP控制蛋白质。在这里介绍的情况下，通过插入一个核定位信号转换成pLuc和pYFP向量生成核针对性吕克和YFP结构。 </li><li>使其中卢克和YFP融合成单个多肽阳性对照构建体。在这里呈现的情况下，产生的阳性对照，其中，YFP编码序列被插入到pLuc矢量。 </li><li>选择一个中性填料质粒被用于均衡的DNA的质量中的转染混合物。使用的质粒不具有真核启动子，因此，将无转录活性的哺乳动物细胞，如细菌克隆载体中。 </li></ol> 2制备的DNA混合料<ol><li>估计所有在第1基于260nm处的吸光度所描述的质粒的浓度（1吸光度单位相当于DNA的50微克/毫升）。通过由650沓乘以碱基对的数量确定每种质粒的分子量。使用浓度和分子量来计算每个质粒制备的摩尔浓度。稀释质粒DNA制备到36 Nm的浓度。这些都将是工作股将用于制备用于转染的DNA的混合物。 </li><li>制备含20微升的水至终​​体积1800纳克填料质粒的对照DNA的组合。 </li><li>制备含有5微升的pLuc控制结构的控制DNA混合物。添加填料质粒带来的总DNA质量1800纳克。加水使最终体积为20微升。 </li><li>制备含有5微升pLuc控制结构和5μlpYFP控制结构的控制DNA混合物。添加填料质粒带来的总DNA质量1800纳克。加水使最终体积为20微升。 </li><li>制备含有5微升阳性对照结构的控制DNA混合物。添加填料质粒带来的总DNA质量1800纳克。加水使最终体积为20微升。 </li><li>准备以下的DNA混合，以测试对于感兴趣，X.蛋白质的同源二聚化对于每一个DNA结合结构5微升的相关pLuc构造和5μl的PY的FP构造。添加填料质粒带来的总DNA质量1800纳克。加水使最终体积为20微升。 A）pLuc控制和pYFP-X B）pLuc-X和pYFP控制 C）pLuc-X和pYFP-X </li><li>准备以下的DNA混合来测试一对感兴趣的蛋白质之间的相互作用，X和Y。对于每一个DNA结合结构5微升的相关pLuc构建和5微升的pYFP的构建。添加填料质粒带来的总DNA质量1800纳克。加水使最终体积为20微升。 A）pLuc控制和pYFP-X B）pLuc-X和pYFP控制 C）pLuc控制和pYFP-Y D）pLuc-Y和pYFP控制 E）pLuc-X和pYFP-Y F）pLuc-Y和pYFP-X </li></ol> 3，转染<ol><li>收获汇合从一个75 厘米烧瓶HEK293细胞。稀释的细胞总数的10％为13 ml培养介质中。免除130微升细胞悬浮液到一个白色的各孔中清澈见底的96孔培养板。培养细胞24小时。 </li><li>计算由乘以3的DNA混合物的数量被转染井的数量。带来的无血清培养基中至室温。制备含6.3μL无血清培养基中，每孔0.18微升转染试剂预混液。涡旋混合并在室温下孵育5分钟。 </li><li>通过加入2微升的DNA混合，以20微升无血清培养基/转染试剂主混合物的制备转染混合物。不要旋涡。在室温下孵育10分钟。 </li><li>转染三口井，每个转染混合物分装6.5微升转染混合物，每孔。培养的细胞用于进一步的36-48小时。 </li></ol> BRET信号4。测量<ol><li>溶解活细胞荧光素酶的底物在34毫克/毫升DMSO中通过涡旋。 </li><li>在基材的稀释介质P稀重组活细胞荧光素酶底物在1:1,000重新加热到37℃。让50微升底物稀释介质，每孔。旋涡混匀。沉淀物可能形成，但不会影响检测干扰。 </li><li>从96孔板中吸出培养基。免除加入50μl稀释的活细胞荧光素酶底物在每孔中。培养细胞至少2小时（最多24个小时）。 </li><li>从96孔板中除去盖子，孵育板10分钟，在室温下的发光计内。 </li><li>从吕克和YFP一个度量发射阱的时间。使用过滤器阻挡波长大于470纳米测量从吕克排放。使用500-600纳米的带通滤波器测量从YFP的发射。集成发射信号在10秒内。 </li></ol> 5。数据分析<ol><li>从3井只收填料质粒的平均吕克排放量读数。从所有其他吕克排放读数减去这个值来产生背景消减吕克排放值。</li><li>从3井只收填料质粒平均YFP的发射读数。从所有其他YFP的发射读数减去这个值来产生扣除背景的YFP的排放值。 </li><li>对于每孔，通过扣除背景的吕克排放值除以扣除背景的YFP的排放值，让裸BRET比例。 </li><li>平均从转染仅pLuc-对照质粒三个孔中的未校正的BRET比率。减去这个值从所有其他未校正的BRET比率，得到校正后的BRET比率。 </li><li>对于每个校正BRET比例的剩余组，平均从3转染的孔中的值，以获得最终的BRET比率。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

融合蛋白表达结构的设计是建立在BRET分析的关键步骤。在这里介绍的实验中，感兴趣的蛋白被融合至萤光素酶或YFP的C-末端。它也是可能的，并且可能是必要的，融合蛋白的荧光素酶/ YFP的N-末端。对于某些蛋白，融合仅可在无论是N-或C-末端，以避免蛋白质结构和功能的破坏所接受。此外，对于跨膜蛋白，其中N和C末端位于不同亚细胞，荧光素酶/ YFP蛋白必须融合到这是在同一货舱的互动合作伙伴?...

既古典联动和下一代人类疾病的测序分析都透出参与了一系列生物途径的蛋白的临床意义。这是通常的情况是，之前，他们在这些研究中鉴定，一直存在的这些蛋白质的生物学作用很小或没有调查。一个卓有成效的途径，开始探索的兴趣蛋白质的生物学功能是识别哪些其他蛋白质它与它的生理范围内进行交互。表征分子网络以这种方式提供了深入了解人类表型相关的生物途径。 最常用的大规模筛选方法确定候选互动合作伙伴的利益蛋白酵母双杂交筛选1和质谱为基础的蛋白质组学2。这些方法可以是非常成功的提示潜在的相互作用的蛋白质，但ARË容易受到假阳性结果。因此，确认确定了酵母双杂交或质谱筛查的交互的需要的交互验证利用第二技术。典型的免疫共沉淀或下拉测定法用于这一目的3。使用这样的技术进行验证的一个缺点是对于细胞裂解，从而破坏了细胞内的条件，可能是维持一定的蛋白相互作用所必需的要求。第二个缺点是软弱或短暂蛋白相互作用可在洗涤步骤被打乱。此外，这些测定要求显著动手的时间，在可同时处理的样本的数目是有限的，并且通常需要试剂和方案的费时的优化。 克服某些与免疫共沉淀实验相关联的问题，有几个实验已经基于荧光和biolum开发inescent蛋白质，可以在活细胞中使用。第一个这样的测定法是基于荧光（或福斯特）共振能量转移（FRET），能量的两种荧光蛋白之间的非辐射转移具有重叠发射光谱和激发光谱4。能量转移的效率很大程度上距离相关，因此观察的FRET现象，要求供体和受体荧光基团是近在咫尺。为了测试两种蛋白质的利益之间的相互作用，一种蛋白质被表达为融合与供体荧光团（通常青色荧光蛋白; CFP）和第二作为融合与受体荧光团（通常黄色荧光蛋白; YFP）。这两种蛋白的利益之间的相互作用可能带来的供体和受体荧光基团足够接近的能量转移发生，这将导致光的发射从YFP的受体相对的CFP供体可衡量的增加。 FRET已经成功地检测蛋白质-蛋白质相互作用中的活细胞4。利用FRET用于检测蛋白质 - 蛋白质相互作用的主要缺点是需要外部照明的供体荧光团的激发。外部照明效果在高背景的发射信号，受不必要的激励和两个供体和受体荧光基团的光漂白。这些效应降低了测定，用于检测蛋白质 - 蛋白质相互作用的灵敏度。 从外部照明克服了高背景问题FRET化验的修改是生物发光共振能量转移（BRET）检测5,6。在BRET系统中的供体荧光团所取代的荧光素酶的酶。因此，能量的受体荧光团的激发在系统内生成的荧光素酶底物的氧化，使外部照明是不必要的。在最该测定的通用结构中，供体是海肾reniformis荧光素酶和受体是YFP（用于替代供体和受体蛋白的讨论见PFLEGER 等人5）。因此，在这种系统中，感兴趣的蛋白融合至萤光素酶和潜在的相互作用蛋白对YFP，或反之亦然。在BRET实验需要加入腔肠素作为底物为荧光素酶。由于腔肠素是细胞渗透性的，因此能够在活细胞中进行BRET分析。然而，本机腔肠素是在水溶液中不稳定，和腔肠素的酶无关的击穿既降低衬底可用于测定中的浓度，并产生自发光，从而降低了荧光素酶活性测量的灵敏度。在活细胞使用BRET已经促进了保护coelenterazines，这是在水溶液中稳定，但受细胞内酯酶裂解的发展穿过细胞膜扩散，以产生电池7内部有源腔肠素后第下列添加底物对细胞表达荧光素酶和YFP融合蛋白，由蛋白质 - 蛋白质相互作用产生的能量转移是通过从荧光素酶和YFP监测排放量化。因为蛋白质的相互作用可直接在活细胞中的多孔板进行监测时，BRET分析构成一个简单的，可扩展的方法，用于检测推定的相互作用是成本和时间效益。 除了验证蛋白质组学筛选研究确定假定的干扰作用，在BRET系统也可以用来从感兴趣的蛋白质前生化和结构的研究而产生的测试候选干扰作用。一旦一个蛋白质 - 蛋白质相互作用的存在已经建立（或者通过使用BRET分析或通过其它技术），存在对于BRET分析为EMP是潜在loyed进一步表征的相互作用。例如，相互作用的区域可通过产生蛋白质的截短形式的映射值，并在特定的相互作用残基的参与可以通过创建点突变来证明。此外，在蛋白质-蛋白质相互作用的翻译后修饰，或小分子（如药物或配体）的调节作用可以调查8-10。 BRET的检测也有一个调查患者的DNA鉴定突变的巨大潜力。在情况下，一个突变的致病作用已经确立，学习使用BRET可以揭示更多关于11表型的分子病因学蛋白-蛋白相互作用的基因突变的影响。由于新一代测序方法的出现，人们越来越普遍的几个潜在损害性突变个体内被发现的，在这种情况下，目前还不清楚这是勒VANT的表型12。在这种情况下，BRET分析可能是在评价突变对蛋白功能的影响有价值的，因此它们对疾病的相关性。

<table><tbody><tr><td>Nanodrop 8000</td><td>Nanodrop</td><td></td><td>Any spectrophotometer capable of reading absorbances at 260 nm will be suitable. Determining the molecular mass of the plasmid is crucial for calculating DNA quantities to be used in transfection mixes.</td></tr><tr><td>96-microwell plates, flat bottom, white</td><td>Greiner Bio One</td><td>655098</td><td>White plates reduce the crosstalk between wells and maximize the sensitivity of luminescence detection. Clear-bottomed wells allow monitoring of cell density. Plates must be suitable for cell culture. If using a top-reading luminometer the plate lid should be taken off.</td></tr><tr><td>Infinite F200Pro plate reader with control software</td><td>TECAN</td><td></td><td>Use the &#039;Blue 1&#039; and &#039;Green 1&#039; filters for luminescence measurement and the filter sets and dichoic mirror for GFP for fluorescence measurement. Any top-reading plate reader with capability of measuring dual-color luminescence and fluorescence is suitable.&amp;nbsp;</td></tr><tr><td>pLuc, pYFP, positive control plasmid</td><td>N/A</td><td>N/A</td><td>Plasmids available from the authors upon request.</td></tr><tr><td>pGEM-3Zf(+)</td><td>Promega</td><td>P2271</td><td>Filler plasmid for equilization of DNA mass in transfection mixes. Any plasmid lacking a eukaryotic promoter would be suitable.</td></tr><tr><td>HEK293 cells</td><td>ECACC</td><td>85120602</td><td>Other cell lines that transfect with reasonable efficiency may be suitable.</td></tr><tr><td>DMEM, high glucose, with phenol red</td><td>Gibco</td><td>41966</td><td>This is the medium used for culturing HEK293 cells. Warm in 37 &amp;deg;C waterbath before use. If using a different cell line, replace the growth medium described here with cell-line specific medium.</td></tr><tr><td>DMEM, high glucose, no phenol red (substrate dilution medium)</td><td>Gibco</td><td>21063</td><td>This is the substrate dilution medium used for dilution of the luciferase substrate (EnduRen) as it does not contain phenol red, which reduces the sensitivity of the assay. Contains HEPES to maintain correct pH during luminescence measurements while cells are out of the CO&lt;sub&gt;2&lt;/sub&gt; incubator. Warm in 37 &amp;deg;C waterbath before use.</td></tr><tr><td>OptiMEM</td><td>Gibco</td><td>31985</td><td>OptiMEM is used for dilution of GeneJuice transfection reagent. Other serum-free media would also be suitable. Warm to room temperature before use.</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10270</td><td>For supplementation of cell culture media at a concentration of 10% v/v.</td></tr><tr><td>GeneJuice transfection reagent</td><td>Novagen</td><td>70967</td><td>If using a cell line other than HEK293, it may be necessary to adjust the ratio of Genejuice transfection reagent to DNA in the transfection mixes. Other transfection reagents may be used. If using an alternative transfection reagent, it may be necessary to optimize the amount of DNA used in the transfection mixes based on manufacturer&#039;s instructions.</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td>Use sterile DMSO that is suitable for tissue culture.&amp;nbsp;</td></tr><tr><td>EnduRen live-cell substrate</td><td>Promega</td><td>E6481</td><td>Reconstitute EnduRen at 34 mg/ml in DMSO. Upon dilution of EnduRen in culture medium a precipitate may form. This will not interfere with the assay. Store reconstituted EnduRen at -20 &amp;deg;C, and avoid multiple freeze-thaw cycles. Ensure that reconstituted EnduRen is completely thawed before diluting it in culture medium.</td></tr></tbody></table>

investigating protein-protein interactions in live cells using bioluminescence resonance energy transfer

基于生物发光共振能量转移（BRET）测定提供一种灵敏和可靠的手段来监测蛋白质 - 蛋白质相互作用中的活细胞。 BRET是能量从一个"供体"萤光素酶的非辐射转移到一个"受体"荧光蛋白。在此测定法中最常用的结构中，供体是海肾reniformis荧光素酶和受体是黄色荧光蛋白（YFP）。因为能量转移的效率很大程度上距离相关，观察的BRET现象，要求供体和受体在靠近。为了测试两种蛋白质在培养的哺乳动物细胞的兴趣之间的相互作用，一种蛋白质被表达为融合与荧光素酶和第二作为融合与YFP。这两种蛋白的利益之间的相互作用可能使供体和受体充分接近能量转移发生。与其他技术相比在调查的蛋白 - 蛋白间相互作用的BRET分析是敏感的，只需要很少的操作时间和一些试剂，并且能够检测这是短暂而微弱的，或者依赖于活细胞内发现的生化环境的相互作用。因此，它是用于确认通过酵母双杂交或质谱蛋白质组学研究提示推定的相互作用的理想方法，此外它是非常适合用于映射相互作用区域，评估的翻译后修饰的蛋白 - 蛋白相互作用的影响，并评估在患者的DNA鉴定的突变的影响。

在BRET实验的原理图示于图2。整个这里介绍的实验中所用的测定法，设置在图3中描绘。从转染的细胞的荧光素酶-YFP融合蛋白的强的BRET信号的检测证实了能量转移是可观察在本实验装置（ 图4）。 我们的研究主要集中在FOXP家庭在大脑发育的转录抑制的作用。影响FOXP2导致言语和语言障碍的一种罕见的，其中最突出的特点是发展语言?...

Watch this Scientific Journal Video about Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer at JoVE.com

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

蛋白质之间的相互作用是十分重要的所有细胞过程。使用生物发光共振能量转移，一对蛋白质之间的相互作用可以在活细胞中，并实时监测。此外，潜在的致病突变的效果进行评估。

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 ...

investigating-protein-protein-interactions-live-cells-using

Research

JoVE Journal

Biology

22.8K 次观看.  Max Planck Institute for Psycholinguistics. 蛋白质之间的相互作用是十分重要的所有细胞过程。使用生物发光共振能量转移，一对蛋白质之间的相互作用可以在活细胞中，并实时监测。此外，潜在的致病突变的效果进行评估。 

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

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

科研

生物化学

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

一种超越BRET步骤:通过荧光激发无限制自发光（FUEL）

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

生物工程

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

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, ...

生物学

使用基于 BRET 的方法在活细胞中进行 CRAF-14-3-3 相互作用分析

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

作者聚焦：整合基于 BRET 的检测和罕见突变分析以破译活细胞中的 RAF 激酶调控

This protocol describes a BRET-based assay for measuring the interactions of the CRAF kinase with 14-3-3 proteins in live cells. The protocol outlines steps for preparing the cells, reading BRET emissions, and data analysis. An example result with identification of appropriate controls and troubleshooting for assay optimization is also presented.

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot ...

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 ...

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

活细菌中蛋白质-蛋白质相互作用的FLIM-FRET测量。

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

评估具有FRET敏化发射的活细胞中的蛋白质相互作用

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

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

摘要

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此视频中的章节

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

一种超越BRET步骤:通过荧光激发无限制自发光（FUEL）

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

成像蛋白质 - 蛋白质相互作用在体内

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

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

活细菌中蛋白质-蛋白质相互作用的FLIM-FRET测量。

评估具有FRET敏化发射的活细胞中的蛋白质相互作用

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

摘要

探索更多视频

此视频中的章节

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

一种超越BRET步骤:通过荧光激发无限制自发光（FUEL）

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

成像蛋白质 - 蛋白质相互作用在体内

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

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

活细菌中蛋白质-蛋白质相互作用的FLIM-FRET测量。

评估具有FRET敏化发射的活细胞中的蛋白质相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用