我们感谢奥布赖恩在UIC实验室关键的实验评价和讨论。这项工作是由美国心脏协会授予11SDG5230003威乐和国家推进转化科学中心 - UIC批准UL1TR000050临床和转化科学中心。

1。细胞转染<ol><li>板细胞转染前一天，电镀免疫细胞较少。 <ol><li>的免疫:在6孔的板，涂层的玻璃盖玻片（1）用0.02％的明胶在37℃下至少4小时，洗一次的介质，并为每个反应孔，板1×10 5 HEK 293T细胞在2毫升的完整的​​生长培养基[Dulbecco改良的Eagle培养基（DMEM）中加10％FBS]。 </li><li>对于流式细胞术和Western blot分析:板1.2×10 5 HEK 293T细胞/孔，在2毫升完全培养基的6孔板（0.3×10 5的HEK 293T细胞在0.5ml完全培养基的24孔板中，每孔）。 </li></ol></li><li>准备用于转染的DNA，根据制造商的协议:Effectene（Qiagen公司）用于免疫荧光。中转-LT1（Mirus公司）用于流式细胞仪和Western blot分析。下面列出了所使用的DNA量。 <table border="1"><tbody><tr><tD&gt; </td><td> 24孔培养板（纳克） </td><td> 6孔板（纳克） </td></tr><tr><td> CFP矢量</td><td> 10 </td><td> 50 </td></tr><tr><td> VN N-AKAP - LBC </td><td> 200 </td><td> 1000 </td></tr><tr><td> VC的N-PDE4D3-FL（PDE4D3全长） </td><td> 2.5，5，10，20，40，80 </td><td> 100 </td></tr><tr><td> VC N-PDE4D3 UCR1（PDE4D3含有UCR1域的N-末端片段） </td><td></td><td> 100 </td></tr><tr><td> VC N-PDE4D3 UCR2 + CAT（PDE4D3 UCR2和催化结构域的C-末端片段） </td><td></td><td> 100 </td></tr></tbody></table>转用（6）Effectene: <ol><li>添加指示的质粒DNA量+ 4微升增强到100微升缓冲EC，混合，在室温下孵育5分钟。 </li><li>添加5微升Effectene，混合，和孵育10分钟室温。添加逐滴细胞。 </li></ol>转公交LT1（6-well/24-well）: <ol><li>指定量的质粒DNA加入到250μl/50微升的Opti-MEM I的无血清培养基的无菌管中。 </li><li>添加过境-LT1试剂的稀释的DNA混合物和吸管轻轻混合。 （过境LT1试剂:DNA比例是3微升过境LT1试剂的每1微克DNA）。 </li><li>在室温下孵育30分钟，并逐滴加入细胞。 </li></ol></li></ol> 2。流式细胞仪，免疫印迹，免疫细胞制备方法<ol><li>染后24小时，检查前收获细胞荧光落射荧光显微镜下。 </li><li>流式细胞仪分析细胞的准备: <ol><li>用0.5毫升/ 2毫升为24-well/6-well板冰冷的PBS洗涤细胞。 </li><li>使用50μl/250微升0.05％胰蛋白酶分离细胞。 </li><li>用200μl/ 1毫升的DMEM培养基中和胰蛋白酶。 </li><li>合作250微升细胞llect（24孔和6孔板），在500×g离心5分钟，通过离心分离，用Western印迹分析的其它细胞（1毫升）。 </li><li>细胞重悬于250微升FACS缓冲[PBS + 0.5％（W / V）BSA + 2毫米EDTA]，储存在冰流式细胞仪分析。细胞也可以固定在1％多聚甲醛（在PBS中），不会立即生效，如果流式细胞分析。 </li></ol></li><li>平行进行的Western印迹分析的细胞制备方法:用流式细胞仪分析细胞制备方法。 <ol><li>用2ml冰冷的PBS 1X洗涤细胞，裂解细胞，加入100μl的细胞裂解缓冲液[10mM磷酸钠缓冲液，pH值为6.95，150 mM氯化钠，5mM的EDTA，5mM的EGTA，1％的Triton X-100] 。 </li><li>通过离心收集细胞裂解液在4℃下以21,000×g的10分钟</li><li>量化Bradford蛋白检测蛋白浓度，装入等量的每通道蛋白SDS-PAGE，并转​​移到硝酸纤维素免疫。 </li></醇&gt; </li><li>免疫细胞制备。 <ol><li>用2ml PBS冲洗细胞3次，在1毫升的3.7％多聚甲醛（在PBS中）在室温下10分钟固定细胞，然后用2毫升的PBS洗涤3次，持续5分钟。 </li><li>挂载，盖玻片载玻片上，用安装介质。如果需要使用指甲油密封盖玻片边缘。 </li><li>将在4℃检查前的幻灯片。 </li></ol></li></ol> 3。流式细胞仪和免疫荧光显微镜<ol><li>使用的Beckman Coulter公司青色ADP，装有488纳米，405纳米，642纳米的固态激光器的流式细胞术进行。峰会软件用于采集和分析。 <ol><li>校准流式细胞仪使用标准的珠子。 </li><li>剧情前向散射（FSC）/侧向散射（SSC）的线性浇注活细胞人口规模。 </li><li>剧情FSC /电压脉冲宽度选通非聚合活细胞群体。 </li><li>剧情count/FL6（CFP fluorescencE，405 nm）的非聚集现场浇注CFP阳性细胞对数刻度。 </li><li>的的剧情count/FL1（YFP的荧光，488 nm）的对数刻度附设强度。 </li><li>运行YFP-CFP-双阴性样品，调整FSC，SSC，FL1和FL6光电倍增管（PMT）电压来设置每个信号的阈值。 </li><li>跑单阳性（两个YFP +和CFP +）样品未来离线补偿</li><li>获得至少10,000门事件（非聚合现场CFP +细胞）进行分析。 </li></ol></li><li>免疫荧光显微镜使用蔡司​​LSM 510共聚焦显微镜注:重要的是要保持所有的设置（如变焦，针孔探测器的增益，放大器的失调，帧大小，扫描速度快，扫描平均，和激光功率）之间的一致样品进行适当比较的所有数据。 </li></ol> 4。数据分析（使用首脑软件执行） <ol><li>设置分析本收类似协议正确的门控离子: 1号门FSC / SSC点图活细胞。 2号门在FSC /脉冲宽度1号门非聚合活细胞中。 3号门，3号门计数/ CFP非聚合CFP +活细胞。 </li><li>补偿YFP和CFP信号的使用YFP +和CFP +单阳性细胞。 </li><li>重新确定切断线CFP / YFP的阳性细胞。 </li><li>进出口YFP的平均荧光强度（MFI）CFP +细胞到Excel的数据绘制。 </li><li>标准化YFP MFI AKAP - LBC，PDE4D3表达水平，并装载控制α-微管蛋白，Western blot检测。 </li></ol>

<ol>
	<li>Collura, V., Boissy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+protein-protein+complexes+to+interactomics.">From protein-protein complexes to interactomics.</a> Subcell Biochem. 43, 135-183 (2007).</li><li>Stumpf, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+size+of+the+human+interactome.">Estimating the size of the human interactome.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6959-6964 (2008).</li><li>Shekhawat, S. S., Ghosh, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-protein+systems:+beyond+binary+protein-protein+interactions.">Split-protein systems: beyond binary protein-protein interactions.</a> Curr. Opin. Chem. Biol. 15, 789-797 (2011).</li><li>Dwane, S., Kiely, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+used+to+study+how+protein+complexes+are+assembled+in+signaling+cascades.">Tools used to study how protein complexes are assembled in signaling cascades.</a> Bioeng. Bugs. 2, 247-259 (2011).</li><li>Piehler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+methodologies+for+measuring+protein+interactions+in+vivo+and+in+vitro.">New methodologies for measuring protein interactions in vivo and in vitro.</a> Curr. Opin. Struct. Biol. 15, 4-14 (2005).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat. Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. J., Suarez, C. D., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET+assay.">Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.</a> Nat. Protoc. 3, 1693-1702 (2008).</li><li>Wong, K. A., O'Bryan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation.">Bimolecular fluorescence complementation.</a> J. Vis. Exp. (50), e2643 (2011).</li><li>Sugarbaker, E. V., Thornthwaite, J. T., Temple, W. T., Ketcham, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+general+principles+and+applications+to+selected+studies+in+tumor+biology.">Flow cytometry: general principles and applications to selected studies in tumor biology.</a> Int. Adv. Surg. Oncol. 2, 125-153 (1979).</li><li>Koshy, S., Alizadeh, P., Timchenko, L. T., Beeton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+GLUT4+translocation+to+the+plasma+membrane+by+flow+cytometry.">Quantitative measurement of GLUT4 translocation to the plasma membrane by flow cytometry.</a> J. Vis. Exp. (45), e2429 (2010).</li><li>Smith, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+confocal+microscopy.">Basic confocal microscopy.</a> Curr. Protoc. Mol. Biol. Chapter 14 (Unit 14), 11 (2008).</li><li>Westwick, J. K., Lamerdin, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+drug+discovery+with+contextual+assays+and+cellular+systems+analysis.">Improving drug discovery with contextual assays and cellular systems analysis.</a> Methods Mol. Biol. 756, 61-73 (2011).</li><li>Kilpatrick, L. E., Holliday, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+pharmacology+of+G+protein-coupled+receptor+signaling+complexes+using+bimolecular+fluorescence+complementation.">Dissecting the pharmacology of G protein-coupled receptor signaling complexes using bimolecular fluorescence complementation.</a> Methods Mol. Biol. 897, 109-138 (2012).</li></ol>

作者宣称，他们有没有竞争经济利益。

附设一个简单而敏感的方法来研究蛋白质相互作用。然而，此方法可以不被使用，以确定新的蛋白质 - 蛋白质相互作用，它是特别方便的，以确认细胞内的蛋白 - 蛋白相互作用和学习功能性质，如细胞内的本地化的蛋白质复合物，蛋白质 - 蛋白质相互作用的网站的映射，并筛查，可以调节蛋白质相互作用的小分子/肽。 ，因为VN和VC片段的非荧光，荧光背景低，从而帮助检测灵敏度。需要时间来成?...

650000蛋白质-蛋白质相互作用，估计存在于人类的相互作用组，发挥着重要的角色，在维持正常的细胞功能1,2。除了 ​​免疫共沉淀（CO-IP）的黄金标准蛋白质相互作用研究从细胞裂解物，一些蛋白质片段互补分析（PCA）已发展到提高灵敏度检测细胞内的蛋白质相互作用3。这些技术包括福斯特共振能量转移（FRET），生物发光共振能量转移（BRET），和双分子荧光互补（附设）4,5。附设是基于荧光蛋白的两个片段（在这里我们使用金星，YFP变种），分别融合到一个潜在的相互作用的蛋白伙伴（在本示例中，我们使用AKAP - LBC和PDE4D3）促进协会。相互作用的两种蛋白质在细胞内的查询结果以功能性荧光的兴趣，它可以是由f可视化luorescence显微镜可以使用活的或固定的细胞6,7,8。相较于其他的PCA，附设敏感的和技术上的挑战，有潜力在活细胞中的细胞定位研究。然而，这种技术的主要缺点是，一旦形成，荧光蛋白复合物不能被逆转。因此，它是不是一个很好的方法来研究动态蛋白质相互作用。在本文中，我们使用附设映射AKAP - LBC PDE4D3的相互作用位点监测的荧光强度金星。相比传统的分析，使用荧光显微镜，这是费时和劳动密集的（如果不进行使用自动化高通过机械），流式细胞术十万个单元格在一个异构的人口提供了一个简单的定量分析，在很短的时间9， 10。在这里，我们开展了流式细胞附设分析和传统的合作IP并排侧比较，表明2米然而，ethods提供可比数据的流式细胞仪附设分析耗时少，并使用更少的材料是有限的感兴趣的细胞时，这可能是更有用的。

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>Dulbecco&amp;#8217;s modified Eagle&amp;#8217;s media (DMEM)</td><td>Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>Penicilin-Streptomycin (pen/strep), 100x</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>Anti-Flag antibody</td><td>Sigma</td><td>M2, F1804</td><td></td></tr><tr><td>Anti-HA antibody</td><td>Covance</td><td>16B12, MMS-101R</td><td></td></tr><tr><td>&amp;#945;-tubulin</td><td>Sigma</td><td>DM1A, T9026</td><td></td></tr><tr><td>Anti-GFP antibody</td><td>Clontech</td><td>632569</td><td></td></tr><tr><td>Cell culture plates, 6-well tissue culture treated</td><td>Thermo Fisher Scientific</td><td>130184</td><td></td></tr><tr><td>HEK 293T cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>Maxiprep plasmid purification kit, high speed</td><td>Qiagen</td><td>12663</td><td></td></tr><tr><td>Dulbecco&amp;#8217;s Phosphate-buffered saline (DPBS), sterile 1x</td><td>Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>Trypsin-EDTA, 0.05% (w/v)</td><td>Gibco</td><td>25300</td><td></td></tr><tr><td>Polystyrene round-bottom tubes for FACS staining</td><td>BD Biosciences</td><td>352052</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>S74337MF</td><td></td></tr><tr><td>Prolong gold antifade reagent with DAPI</td><td>Invitrogen</td><td>P-36931</td><td></td></tr><tr><td>Superfrost plus Microscope slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Fisherfinest premium cover glass</td><td>Fisher Scientific</td><td>12-548-5P</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; air-jacketed Incubator</td><td>NuAIR DH autoflow</td><td></td><td></td></tr><tr><td>Confocal microscope LSM510 META</td><td>Carl Zeiss, Inc</td><td></td><td></td></tr><tr><td>Electrophoresis and transfer unit</td><td>Biorad</td><td></td><td></td></tr><tr><td>Cyan ADP Flow Cytometer</td><td>Beckman Coulter</td><td></td><td></td></tr></tbody></table>

flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction

其中的方法来研究细胞内蛋白质相互作用，双分子荧光互补（附设）是相对简单和敏感。附设是根据生产的荧光使用两个非荧光的荧光蛋白的片段（金星，黄色荧光蛋白的变型中，这里使用的是）。非荧光金星片段的（VN和VC）的两个相互作用的蛋白质（在这种情况下，AKAP-LBC和PDE4D3）稠合，得到的荧光由于VN-AKAP-LBC-VC-的PDE4D3相互作用和形成荧光蛋白的功能细胞内。 附设提供的蛋白质复合物的亚细胞定位和蛋白质相互作用的强度，根据荧光强度的信息。但是，附设分析用显微镜量化蛋白 - 蛋白相互作用的强度是耗时且有些主观的，因为蛋白表达的异质性和相互作用。通过耦合流式细胞仪分析与附设的方法，荧光附设的蛋白 - 蛋白相互作用的信号可以精确地测量在短时间内大量的细胞。在这里，我们展示了这种方法的应用程序AKAP-LBC的互动所需要的地区PDE4D3映射。这种高的通过方法可以应用于筛选因子，调节蛋白 - 蛋白相互作用。

AKAP-LBC和PDE4D3的结构（ 图1B）的融合VN和VC片段，分别AKAP - LBC和PDE4D3结果功能YFP的荧光（ 图1A）的相互作用。在这里，我们使用地图AKAP - LBC结合位点PDE4D3附设的方法。 PDE4家族蛋白中上游保守区域1（UCR1），，上游保守区域2（UCR2）和催化区（CAT）是保守的，因此，全长度（FL），UCR1和UCR2加CAT被用于筛选结合部位的PDE4D3 AKAP-LBC。 VN-AKAP-LBC和VC-PDE4D3片段在HEK 293T细胞荧光结?...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction at JoVE.com

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

流式细胞仪分析双分子荧光互补提供了一种高通量定量研究蛋白质相互作用的方法。此方法可用于测绘的蛋白结合位点，并用于筛选调节蛋白 - 蛋白相互作用的因素。

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 ...

flow-cytometric-analysis-bimolecular-fluorescence-complementation

Research

JoVE Journal

Biology

18.3K 次观看.  University of Illinois at Chicago. 流式细胞仪分析双分子荧光互补提供了一种高通量定量研究蛋白质相互作用的方法。此方法可用于测绘的蛋白结合位点，并用于筛选调节蛋白 - 蛋白相互作用的因素。

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

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

科研

生物化学

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

光敏定位显微镜与双分子荧光互补（附设-PALM）

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

生物工程

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

发育生物学

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

双分子荧光互补

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

生物学

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

免疫与感染

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

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 ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

通过流动细胞测定检测和描述 Vivo 中的蛋白质自组装

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

FLIM-FRET Imaging for Characterization of Protein-Protein Interactions in Live Bacteria

Source: Manko, H. et al., <a href="https://app.jove.com/v/61602">FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.</a> J. Vis. Exp. (2020)
The video describes the FLIM-FRET imaging technique to determine the protein-protein interaction in live bacteria expressing cytoplasmic proteins labeled with fluorescent proteins, a donor eGFP, and acceptor mCherry. The combined technique also allows the quantification of the interacting proteins.

用于表征活细菌中蛋白质-蛋白质相互作用的 FLIM-FRET 成像

Source: Manko, H. et al., FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria. J. Vis. Exp. (2020)
The video describes the FLIM-FRET ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy

Source: Nickerson, A. et al., <a href="https://app.jove.com/v/53154">Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM).</a> J. Vis. Exp. (2015)
This video describes BiFC-PALM &#8213; a combination of photoactivated localization microscopy and bimolecular fluorescence complementation &#8213; to assess protein-protein interactions. BiFC involves the fusion of two fluorescent protein fragments with two interacting proteins of interest. When the two proteins interact, the fragments are brought into proximity, which allows the two parts to come together and reconstitute a functional fluorescent protein. The fluorescence of a single fluorophore is localized at a single molecule level inside cells by PALM.

双分子荧光互补耦合光激活定位显微镜

Source: Nickerson, A. et al., Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM). J. Vis. Exp. (2015)
This ...