我们要感谢于尔根索尔的有益讨论和克里斯嘉莉的手稿的批判性阅读。该项目资助的东风集团和全宗DER chemischen工业公司（补助号SFB 1035项目A04至SS和做二十二分之一百八十七到RS）通过。

1。附设的转型构建农杆菌中<ol><li>附设构建的克隆<ol><li>使用含侧翼attB位站点寡核苷酸相应的模板扩增目的基因。执行使用校对聚合酶的PCR。适应的退火步骤来设计引物组合的长度，并根据片段大小的延伸步骤的长度。检查PCR产物通过琼脂糖凝胶电泳，并通过使用PCR清洁试剂盒纯化它。 </li><li>执行与所获得的片段即BP反应，并用BP重组酶的入门载体。混合15-150纳克的attB位-PCR产物用150纳克的条目的向量，加入2微升的BP重组酶，并用TE缓冲液补满至8微升的总体积。在室温下孵育1小时，进行反应和终止反应，加入2微升蛋白酶K 10分钟，在37℃下</li><li>改变整个反应到感受态大肠杆菌 DH5α;细胞并筛选所得到的菌落PCR克隆为正确的插入的DNA片段插入载体中。的阳性质粒DNA测序进行验证的情况下的突变。 </li><li>使用LR重组酶使用获得入门克隆进行LR重组与相应的目标向量。搭配50-150纳克入门载体与150毫微克的目标向量，加1微升的LR重组酶，并用TE缓冲液填补高达5微升的总体积。在室温下孵育1小时，进行反应和终止反应，加入1微升蛋白酶K 10分钟，在37℃下</li><li>改变整个反应成通过菌落PCR对正确插入的DNA片段到载体中获得的菌落感受态大肠杆菌DH5α细胞和屏幕。 DNA测序是没有必要在这一步。 </li><li>分离质粒DNA，用质粒Mini试剂盒，以确保纯度的高度。 </li></ol></li><li>化学感受态农杆菌（制备菌株AGL1，利福平和羧苄青霉素抗性） <ol><li>从A股文化连胜了农杆菌，并在28℃下生长24小时</li><li>接种5ml的LB培养基中单个菌落孵育过夜，在28°C。 </li><li>接种50毫升LB培养基与2ml的过夜培养物，并在28℃生长约4小时，至OD 600为1.0。 </li><li>离心将细胞以3,000 xg离心15分钟，在4℃，重悬沉淀于1ml灭菌的冰冷的CaCl 2（10毫米）。这一步后，细胞置于冰上。 </li><li>制备的细胞的等分试样（100微升），在液氮中，并储存在-80℃下立即冷冻</li></ol></li><li>化学感受态农杆菌转化<ol><li>在冰上解冻胜任AGL1细胞的一个等分。加1-2微克质粒DNA到细胞中。孵育5分钟，在冰上5分钟，在液氮中和5分钟，在37℃下添加600μl的LB培养基中至细胞和s鳕鱼在650转4小时，在28°C。 </li><li>离心细胞1分钟，在8000×g离心并弃去上清液。重悬沉淀中加入50μl的剩余的LB培养基和板在含有适当抗生素的LB平板上的。密封板，并培育2天在28°C。屏幕菌落的质粒通过菌落PCR的存在。 </li><li>在含有适当抗生素5ml的LB培养基中接种一个正殖民地和孵化在28℃下过夜。混合500微升过夜培养物用500μl的50％无菌甘油冷冻于-80℃。 </li><li>接种在LB培养基中含有直接从甘油库存烟叶瞬时转化合适的抗生素农杆菌。 </li></ol></li></ol> 2。烟叶瞬时转化<ol><li>农杆菌增长<ol><li>准备下列储备液：乙酰丁香酮（150毫米，溶于70％乙醇），存储作为分装于-20°C。 MES / KOH（0.1M），pH值5.7，在4℃（通过0.2微米的过滤器，以防止在较长的储存细菌生长无菌过滤器）店， 氯化镁 （1米）的股票，在室温下储存。 </li><li>接种含有适当抗生素用50μl含在无菌50ml管中感兴趣的质粒中的AGL1甘油储用培养10毫升LB培养基中，孵育在28℃下至少24小时，振摇，在190转，直到OD 600为1.0 -2.0为止。 </li><li>离心机菌在3000×g离心15分钟。重悬沉淀在新鲜的浸润培养基[MgCl 2的（10毫米），MES / KOH（10毫米），pH值5.7，乙酰丁香酮（150μM）]和调整悬浮液的OD 600为1.0。 </li><li>在孵育的开销摇床2小时在黑暗的农杆菌细胞。细胞然后可以用于浸润。 </li></ol></li><li>烟叶渗透<ol><li>使用三个周龄烟草（ 烟草本塞姆氏 ）植物。选择几种老叶渗透。 </li><li>混合携带感兴趣的构建体（各3ml）中的农杆菌等体积。取5毫升的无针注射器进行渗透。通过按压在叶片上的底部侧的注射器在几个地方仔细渗入细胞悬液到烟草叶子。 </li><li>给植物浇水，让他们在黑暗中两天。 </li></ol></li></ol> 3。原生质体的制备烟叶原生质体的制备是改编自库普等人 16和稍作修改。 <ol><li>缓冲液配制<ol><li>准备的F-PCN介质：宏观盐[KNO 3（1012微克/毫升），氯化钙2•2H 2 O（440微克/毫升），硫酸镁4•7H 2 O（370微克/毫升），KH 2 PO 4（ 170微克/毫升），NH4-琥珀酸酯[（20毫米;准备一个2M的股票溶胶ution（琥珀酸（236微克/毫升）与NH 4 Cl（106微克/毫升）中，调节pH至5.8以溶解）]，微盐[EDTA-铁（III）×钠-盐（40微克/毫升）， KJ（0.75微克/毫升），H 3 BO 3（3微克/毫升），硫酸锰4•H 2 O的（10微克/毫升），硫酸锌4•7H 2 O（2微克/毫升），钠2的MoO 4• 7H 2 O（0.25微克/毫升），硫酸铜4•5H 2 O（0.025微克/毫升），氯化钴2•6H 2 O（0.025微克/毫升）]，MES（390微克/毫升），葡萄糖（约80微克/ ml）的渗透压550毫渗透摩尔浓度，pH值5.8（KOH）。在储存分装于-20°C。 </li><li>准备的F-PIN介质：所有成分为葡萄糖，而不是使用蔗糖（约110微克/毫升），渗透压550毫渗量，pH值5.8（KOH）的F-PCN但。在储存分装于-20°C。 </li><li>准备W5介质：150 mM氯化钠，125毫米氯化钙2，5毫米氯化钾，2毫米的MES，渗透压550-580毫渗量，pH值5.7（KOH）。储存于4°C（通过0.2μm过滤器无菌过滤器，以防止在较长的储存细菌生长）。 </li><li>准备原生质体分离（0.1克纤维素酶，在10毫升的F-PIN0.03克macerozym）新鲜酶液。孵育的溶液在55℃下进行10分钟并冷却至室温。加入100微升10％的BSA至10ml溶液。 </li></ol></li><li>原生质体的分离<ol><li>将一个叶片渗透到培养皿，加入新鲜酶液。使用新的剃刀刃切叶成大约0.5 平方厘米大小的块。传输叶片与酶溶液放入真空渗透烧瓶和真空渗入约20秒，直到气泡从叶子出现（释放真空非常小心）。 </li><li>振摇90分钟，40转在黑暗中。 </li><li>在90 rpm振摇1分钟释放原生质体。通过纱布（100μM）过滤该溶液到15毫升的离心管（圆底）。 </li&gt; <li>叠加后的原生质体溶液与2ml的F-PCN缓冲，离心，在70×g离心（慢加速和减速），在室温下10分钟。 </li><li>完整的原生质体聚集在酶溶液和F-PCN接口。采取多种孔板的1毫升吸管尖端完整的原生质体转移到一个新的离心管中，并填写了W5的缓冲区。离心2分钟，在100 XG（缓慢加速和减速），以沉淀原生质体。 </li><li>通过使用在约200微升W5缓冲吸管和重悬沉淀，这取决于原生质体的量小心取出上清液。 </li><li>始终使用宽口提示，以防止完好的原生质体破裂。 </li></ol></li></ol> 4。激光扫描显微镜<ol><li>样品制备<ol><li>粘贴密封胶围绕一个载玻片两小片（相距2厘米）。将20微升的带之间的原生质体溶液，并小心地将玻璃盖在上面。密封条确保原生质体没有被盖玻璃被挤压。 </li><li>对总叶样品切成1厘米的片从叶，并将其放置到与朝上叶子的底部侧的显微镜载玻片。加入约30微升H 2 O，放置在顶部玻璃盖和两侧胶带紧紧固定。 </li></ol></li><li>共聚焦成像和显微设置<ol><li> TCS SP5：成像与徕卡，类型共聚焦激光扫描显微镜进行。对于使用放大倍数的物镜（HCX PL APO CS）与63X幅度以甘油为成像介质。设定的数值孔径至1.3。使用徕卡应用套件/高级荧光软件进行评价（见补充资料S1）。 </li><li>在488nm处设置的氩激光到30％，而激光功率的18％的强度来监控重构附设信号在515 nm和从495-550设为1的PMT探测器发射带宽。 </l我&gt; <li>为了监测叶绿素自发荧光从650-705集第二PMT探测器发射带宽。 </li><li>来监视的mCherry信号使用氦氖激光器561中，设置激光的强度561〜18％，从587-610三分之一PMT检测器的发射带宽。 </li><li>确保所有的光电倍增管检测器通道的照片是用相同的增益设置（增益应该是800-900之间排除背景信号）。 </li><li>拍照1024 x 1024像素，100赫兹的扫描速度的幅面宽度/高度。 </li><li>为Z型零件组使用的每个协议栈之间0.5μm的最大距离。 </li></ol></li></ol>

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作者什么都没有透露。

当规划一个附设实验几点应予以考虑。虽然对感兴趣的蛋白质没有结构信息是必需的，拓扑与跨膜蛋白质工作时是已知的。荧光蛋白必须驻留在同一个亚细胞区室或面对的膜，以允许相互作用的同一侧。自然地，分析这需要一个N-末端的定位序列的蛋白质时，只有一个C-末端标签可以考虑的。因为它是可能的标记干扰感兴趣的蛋白质，最好是预先测试的亚细胞定位，例如，通过表达GFP标记的蛋白的...

研究蛋白质复合物的形成和它们在植物细胞在体内的定位是必不可少的探讨蜂窝网络，信号和代谢过程。附设允许的蛋白质-蛋白质相互作用可视化直接在活体植物细胞1-5在他们的自然环境。 在附设接近的荧光蛋白引到一个重组荧光蛋白2非荧光性的N-和C-末端片段的互补。许多不同的荧光蛋白的片段已被用于检测蛋白质相互作用， 例如绿色荧光蛋白（GFP），其中由三种不同的残基6化学键形成的发色团。荧光蛋白可以在循环或β-链以产生两个非荧光片段可以被融合到感兴趣的两种蛋白质内减半。该测定可用于检测任何亚细胞compartme相互作用nt的任何需氧生长的生物体或细胞，可以进行基因修饰以表达融合蛋白。如果这两种蛋白进入细胞内的接近，荧光被重构，并且可以通过显微镜，而不添加外源荧光团或染料3来监控。 烟草（ 本氏烟 ）已被证明是方便的模型有机体可视化植物蛋白质的相互作用，因为蛋白质可以容易地通过利用烟叶与所产生的构建体农杆菌介导的转化来表达。农杆菌使用一个所谓的Ti质粒（肿瘤诱导）编码介导感兴趣的基因转导到植物细胞中的酶。附设是很好适用于水溶性以及对所有细胞区室中的膜蛋白，并已成功地用于在过去几年，以确定在体内相互作用的蛋白质，以及对分析相互作用位点内蛋白质7-9。待导入的基因表达，荧光蛋白的相互作用，可以直接在叶片可视化，这是适合于较大的蜂窝结构，如内质网（ER），在质膜或叶绿体。然而，为了监视在更精细的结构定位，例如，叶绿体被膜，最好是可视化的荧光在原生质体从转化的烟草叶中分离。一组含有任一C-末端或N-末端的荧光标记有用于在植物10的附设方法附设载体。在下文中描述的协议被用来研究细胞内的热休克蛋白90（HSP90）的相互作用与三十四肽重复含蛋白质对接Toc64和AtTPR7居住在叶绿体外封套和内质网，分别11-13（TPR）结构域。为了这个目的，HSP90融合到的CtSCFP（SCFP C）的erminal一部分。标签上还有N端融合到伴侣，以确保HSP90的C-末端MEEVD结合基序钳型TPR结构域的可访问性。与此同时，金星（金星N）的N-末端部分融合到含有蛋白质对接Toc64和AtTPR7分别TPR结构域的胞内域。作为阴性对照，我们克隆SCFP 的C可溶性C-末端部分完全驻留在细胞质中，因此适当的控制。 所研究的蛋白质的荧光标记要面对同样的细胞区室，让靠近，从而荧光信号的重建。来确定重构的荧光信号稠合到不同荧光标签标记的蛋白质可共转化来演示的相互作用的亚细胞定位的定位。融合到mCherrry一个ER标志物蛋白的同时转化案例位于AtTPR7 14急诊室的。叶绿素的自发荧光担任叶绿体标记的情况下Toc64的。通过这不仅Toc64和AtTPR7，分别与胞质HSP90分子伴侣的体内相互作用，可以直接在烟草叶片监测，但也相互作用的亚细胞定位可以进行调查。 附设很适合作为一种补充的方法来研究蛋白质 - 蛋白质相互作用的其它方法。相比于免疫共沉淀或体外下拉实验，例如，没有特异性抗体必须是可用于感兴趣的蛋白质，以及这些蛋白质没有被重组表达在体外 ，它可以是具有挑战性的，特别是对于膜蛋白。此外，也瞬时相互作用可以利用附设监视，因为蛋白质是由稠合的荧光标签15的相互作用捕获。

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		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">3&#39;,5&#39;-Dimethoxy-4&#39;-hydroxyacetophenone</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:255px;">D134406</td>
			<td style="width:195px;">Acetosyringone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Cellulase, Onozuka-R10</td>
			<td style="width:147px;">Serva</td>
			<td style="width:255px;">16419</td>
			<td>from Trichoderma viridae</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Macerozyme R-10&#160;</td>
			<td style="width:147px;">Serva</td>
			<td style="width:255px;">28302</td>
			<td>from Rhizopus sp</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:293px;">GATEWAY, BP Clonase II, Enzyme Kit</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:255px;">11789-(020)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">GATEWAY, LR Clonase II, Enzyme Kit</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:255px;">11791-(020)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:255px;">27106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">NucleoSpin Gel and PCR Clean-up Kit</td>
			<td style="width:147px;">Macherey-Nagel</td>
			<td style="width:255px;">740609-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:293px;">pDEST-GWVYNE</td>
			<td style="width:147px;"></td>
			<td style="width:255px;">Gehl C. et al, 2009, Molecular Plant</td>
			<td>Gateway-cloning (Invitrogen)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:293px;">pDEST-VYNE(R)GW</td>
			<td style="width:147px;"></td>
			<td style="width:255px;">Gehl C. et al, 2009, Molecular Plant</td>
			<td>Gateway-cloning (Invitrogen)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:293px;">pDEST-SCYCE(R)GW</td>
			<td style="width:147px;"></td>
			<td style="width:255px;">Gehl C. et al, 2009, Molecular Plant</td>
			<td>Gateway-cloning (Invitrogen)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;"></td>
			<td style="width:147px;"></td>
			<td style="width:255px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

protein-protein interactions visualized by bimolecular fluorescence complementation in tobacco protoplasts and leaves

许多蛋白质​​相互作用的瞬时与其他蛋白质或集成到多蛋白复合物以发挥其生物学功能。双分子荧光互补（附设）是一种体内方法来监控在植物细胞中这种相互作用。在所提出的协议所研究的候选蛋白融合至荧光蛋白互补的两半，并在各自的结构是通过农杆菌介导的转化导入植物细胞。随后，将蛋白质瞬时表达在烟草叶和还原的的荧光信号可以在完整的细胞共聚焦激光扫描显微镜来检测。这允许相互作用本身不仅可视化，而且还蛋白复合物的亚细胞定位可以被确定。为此目的，含有荧光标记的标记基因可以一起附设构建体共表达，从而可视化的细胞结构，如叔他面内质网，线粒体，高尔基体或质膜。荧光信号可以直接在表皮叶细胞或单原生质体，可从转化的烟草叶容易地分离出被监控。附设非常适合于活细胞内研究在其自然环境中的蛋白质 - 蛋白质相互作用。然而，它必须被认为表达必须通过强启动子来驱动，而相互作用伙伴由于融合了相对大的荧光标记，这可能与交互机制干扰的修改。然而，附设是一个很好的补充方法，其它常用的方法研究蛋白质-蛋白质相互作用，如免疫共沉淀， 体外下拉测定法或酵母双杂交实验。

在这个例子中，我们使用了附设法来监测细胞内分子伴侣的HSP90的膜对接蛋白AtTPR7和Toc64的相互作用。 AtTPR7是二段易位子的一部分，并与细胞内分子伴侣，其可能传送分泌前蛋白进行翻译后转运到ER膜相互作用。同样，Toc64在叶绿体外膜由接收HSP90相关的叶绿体前蛋白的作用在翻译后导入。两种蛋白包括细胞内露出TPR结构域，其介导的相互作用与HSP90的C-末端MEEVD动机。 蛋白通过...

Watch this Scientific Journal Video about Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves at JoVE.com

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

蛋白质复合物在体内的形成可以通过双分子荧光互补可视化。合作伙伴的互动融合，以荧光标记的互补部件和瞬时表达烟叶，导致后两种蛋白质的接近改组后的荧光信号。

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 ...

protein-protein-interactions-visualized-bimolecular-fluorescence

Research

JoVE Journal

Biology

20.7K 次观看.  Ludwig-Maximilians-Universität, München. 蛋白质复合物在体内的形成可以通过双分子荧光互补可视化。合作伙伴的互动融合，以荧光标记的互补部件和瞬时表达烟叶，导致后两种蛋白质的接近改组后的荧光信号。 

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

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

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 ...

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 ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

荧光显微镜下细胞电融合的可视化

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

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, ...

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 ...

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 ...

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 ...

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 ...

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 ...