We wish to thank the Calcium Imaging Core Facility at CMS for their help and guidance in image capture and analysis. This study was supported by the U.S. National Institutes of Health (NIH) HL102208 to N.A.B. This approach was originally pioneered by Holger Lorenz and Jennifer Lippincott-Schwartz at NIH.

1.代荧光蛋白嵌合体和验证<ol><li>附加绿色荧光蛋白（GFP）与感兴趣使用标准的重组DNA策略13中的蛋白质的氨基或羧基末端。 注意：虽然我们已经使用GFP的广泛应用，其他变型如青色荧光蛋白（CFP），黄色荧光蛋白（YFP），红色荧光蛋白和mCherry可以使用。正在不断取得荧光折叠效率，亮度和光稳定性的改进，和调查应利用最新一代的结构本身。 <ol><li>确认正确的DNA序列，并确保将GFP部分是在框架与感兴趣的蛋白质。使用标准的重组DNA策略21进行测序。 注：很多机构都核心DNA设施，研究者鼓励联系以获取更多信息。 </li><li>对于单程或双程膜蛋白，产生两个氨基和羧基末端的版本融合蛋白19的。 </li><li>对于多生成树蛋白插入推定膜外循环内的GFP，以及末端。产生跨膜嵌合体与附着于靶蛋白的氨基末端的荧光融合蛋白时作为信号序列被蛋白水解裂解通常一旦该蛋白已经通过对ER位子传递特别小心。 </li><li>在现存的信号序列的情况下，插入荧光融合蛋白远端切割位点，以便产生含有完整的氨基末端标记的荧光蛋白的成熟蛋白质。使用用于嵌合体21的生成标准的重组DNA的策略。 </li></ol></li><li>确定足以适当地本地化的荧光信号在感兴趣的细胞系的存在。 <ol><li>使用下appropri GFP的固有荧光观察下荧光显微镜的细胞吃了过滤器设置的条件。对于包括510-560纳米的GFP过滤器设置，确认正确的过滤器用于采用的荧光。 注：足够的信号达到评估时，用肉眼提供了足够强的信号，该信号驻留在其中的细胞结构很容易上述背景下解决。此外，绿色荧光蛋白的染色模式应该是与该标记相应的细胞器的一致，并优选相同的，天然的未标记的蛋白质。 </li></ol></li><li>确定是否基于所述蛋白被评估稳定或瞬时转染的方法将被使用。 注：稳定转染通常得到的表达水平低于瞬时系统。虽然瞬时表达产生的蛋白和增加信号强度（〜5-24小时转染后视蛋白）的高水平表达，这也可导致过度的工件，如蛋白质AGGRegation或蛋白靶向通路的饱和，从而导致错误的亚细胞定位。 </li></ol> 2.转染细胞和细胞培养的注：多种细胞是适合于FPP分析，包括HEK293，HeLa细胞，COS-7和CHO细胞14。下面的描述是基于表达膜蛋白在HEK293细胞。 <ol><li>生长的人胚胎肾细胞（的Hek293）以贴壁单层在6ml的Dulbecco氏改良的Eagles培养基（DMEM）中，在37℃补充有5％胎牛血清（FBS），1％丙酮酸钠，和250微克/毫升青霉素/链霉素下用5％的CO 2。执行在层流组织培养罩的所有工作用的细胞。生长的细胞在培养基中直到〜的T-75烧瓶中80％汇合。 </li><li>使用任何标准的高效率，低毒性的转染方法，包括脂质，viral-或电为基础的技术，以产生transien转染细胞吨或稳定表达。通常为2微克每1×10 6个细胞的质粒DNA给出了良好的重现性蛋白表达。保持在CO 2能够组织培养箱细胞为5-48小时在适当的组织培养基中，在37℃。 </li><li>监测细胞使用荧光显微镜的GFP融合蛋白的表达，采用的GFP的固有荧光信号，根据制造商的协议使用适当的过滤器集。荧光信号通常达到内24-72小时转染后一最大值。使用的细胞时，荧光信号达到最大电平。监测蛋白表达的免疫印迹分析使用针对感兴趣的任一​​蛋白或GFP的部分的抗体的水平。 </li><li>实验分析板细胞上涂盖玻片。查看或者下一个活细胞配置或以下的固定和安装在显微镜幻灯片。板的细胞来实现在24-48小时的60％-80％汇合。 <ol><li>外套盖玻片用聚赖氨酸溶液（0.1毫克/毫升），这样整个表面被覆盖。孵育下紫外光在室温搅拌30分钟，随后在细胞培养孵化器60分钟，在37℃。去除多余的多聚赖氨酸和冲洗的PBS 3倍的盖玻片。 </li><li>替代地，盘上的腔盖玻璃组成的细胞培养板用光学玻璃底的细胞，以允许直接成像油浸目标。在实际的显微镜设置取决于可用的研究者在设备上，但应能迅速解决的变化。根据细胞类型和细胞形状，细胞应为约60-90％汇合的FPP分析的时间。 </li></ol></li></ol> 3.荧光显微镜安装<ol><li>使用的最大信号采集和空间分辨率，如60X，1.42 NA油浸物镜高数值孔径（NA）油浸物镜。使用日Ë以下激光器激发荧光团：氩气488纳米（GFP和YFP）和561二极管（RFP，mCherry）。 </li><li>确定图像的每个结构的最佳曝光，增益和捕获率。调节激光的强度和照射时间，以减少光漂白（见步骤7.1.2）。 </li><li>用于使用多个荧光团（ 例如 ，可溶性标记在音乐会的膜蛋白标志物膜通透性），选择适当的过滤器的研究设置以最小化信号的串扰和提高信号噪声比。 </li></ol> 4.建立最佳条件等离子膜通透性<ol><li>除去从细胞中只表达可溶性的荧光蛋白质的细胞培养基（ 例如绿色荧光蛋白，红色荧光蛋白）。在KHM缓冲洗细胞3次（每次1分钟）（110毫醋酸钾，3mM的MgCl 2的，20毫米的HEPES-氢氧化钠，pH为7.2）。在室温下执行与细胞的实验和所有试剂。 </li><li>地方细胞ØÑ​​盖玻片（参见步骤2.4）上的荧光显微镜阶段KHM缓冲器，并收集第一图像，其表示该控制“非透化”阶段。 </li><li>以选择性地透质膜，在KHM缓冲器添加毛地黄皂苷（30μM）细胞中。灌注细胞用缓冲液（缓冲液+毛地黄皂苷）中/速率为5毫升分钟为10-60秒。 </li><li>通过逐渐增加毛地黄皂苷的浓度确定最佳的毛地黄皂苷浓度。当有荧光信号的内10-60秒毛地黄皂苷申请完全丧失从可溶性EGFP最优毛地黄皂苷浓度达到。对于许多细胞应用的10-50微米毛地黄皂苷是足够通透的细胞膜。 <ol><li>对于所有的应用程序都使用毛地黄皂苷的尽可能低的浓度。毛地黄皂苷透了透化背景信号后，采集图像。 </li></ol></li><li>确认感兴趣的GFP蛋白嵌合体是膜在通过确认蜂窝荧光信号的保留以下质膜16的毛地黄皂苷透化tegrated。 <ol><li>表达一个特定的细胞器荧光蛋白23。线粒体蛋白质在这方面16， 例如 ，pDsRed2-三刀载体很好地工作。决定按照步骤2.1中所述的最佳表达条件。 </li><li>确认毛地黄皂苷浓度是适合于通过确保所使用的细胞类型的细胞内细胞器的形态保持不变通过长时间（最多60分钟）暴露于毛地黄皂苷。 注意：使用抗体对各种细胞区室用标准免疫荧光显微镜的协议，如溶酶体（ 例如 ，LAMP1），高尔基（ 例如 ，TGN38），内质网（ 例如 ，钙联接蛋白），内体（ 例如 ，Rab5的），线粒体（ 例如 ，细胞色素C）。如果细胞内的细胞器形态/完整性急剧变化奥雅纳R 60分钟，它是可能的毛地黄皂苷浓度过高，并且需要在毛地黄皂苷浓度和/或曝光时间的减少。 注：毛地黄皂苷是吸入和皮肤接触有毒。穿戴合适的个人防护装备（PPE）。 </li></ol></li></ol>荧光蛋白5.蛋白酶处理<ol><li>洗细胞中KHM缓冲，然后添加蛋白酶（蛋白酶K;在KHM缓冲器50微克/毫升）直接到细胞上。灌注细胞以5ml使用重力给料的灌注系统连续浴中的制剂/分钟的流速。 <ol><li>在50毫升的注射器商店的解决方案和聚乙烯管连接独立储到歧管与在浴（5毫升/分钟流速）的单一出口。定位的吸入吸管以保持恒定的（0.5毫升）浴体积和通过灌注容器之间手动切换实现溶液交换。 </li></ol></li><li>收集图像，以确定是否所述氟escent信号持续或降级。 &gt; 90％，比30-60秒的初始信号强度的损失是与GFP蛋白嵌合体的蛋白水解降解相一致（见代表结果）。 </li><li>如果没有信号衰减是用蛋白酶K观察到的，当预计这种损失，取代蛋白酶K溶液用胰蛋白酶（4-8毫米）中KHM缓冲器。如果需要的话，利用蛋白酶K和胰蛋白酶的混合物。没有必要以淬灭蛋白酶活性。 </li></ol>对于质膜蛋白质与外部域6.替代方法<ol><li>对于等离子的膜蛋白，进行检测的，以洋地黄通透前外部荧光蛋白嵌合体。洗在KHM缓冲器盖玻片的细胞（5毫升/分钟×3分钟），并采取预蛋白酶处理的图像和量化的预蛋白酶荧光信号，如步骤7。 </li><li>细胞暴露于蛋白酶（蛋白酶K; 50微克/毫升胰蛋白酶或4-8毫米KHM缓冲液中）在叔他通过灌注细胞KHM含蛋白酶的媒体（流速5毫升/分钟）没有毛地黄皂苷的。收集荧光蛋白的图像，以确定如何快速的信号消失要不了多久，信号仍然存在。 </li><li>毛地黄皂苷透化前，淬火所有蛋白酶活性通过在含有10％血清（FBS）的细胞培养基洗涤细胞三次（每次1分钟）。 </li><li>通过用KHM缓冲液（5毫升/分钟流速）进行2分钟灌注洗细胞。获得的预通透图像。透化细胞膜如步骤4中所述。 </li><li>如在步骤5中所述收集下列蛋白酶加成图像。 </li></ol> 7.图像分析<ol><li>使用倒置显微镜能够“活细胞”的记录，其中，荧光信号的损耗可以实时监测。连续监测，采用相同的参数（曝光时间，增益，激光强度等 ）。 <ol><li>确定EXPERimental参数，通过观察从控制条件（ 即 KBH单独的缓冲液）下感兴趣的GFP蛋白的荧光信号。调整在计算机驱动的显微镜的增益，激光强度和曝光时间设置，以确保有信号强度没有明显的损失超过10-20分钟的时间。 注意：信号强度不是一个绝对值，而是基于显微镜系统和设置可用于每个PI上。监测变化在相对荧光强度。 </li></ol></li><li>此外，治疗的细胞在设定的时间点洋地黄其次是蛋白酶，并修复细胞安装在盖玻片成像之前。确定时间点凭经验按照步骤4和5。初步调查程序，从0开始，30，60，120和300秒后添加洋地黄或蛋白酶。 <ol><li>固定用的固定剂的足量（在PBS中3.7％的多聚甲醛），以充分淹没的Th细胞（20分钟室温）E细胞。 </li><li>通过用含有20mM甘氨酸（3×5分钟）的PBS漂洗细胞淬灭残余的多聚甲醛。 </li><li>摩盖玻片通过从盖玻片除去多余的液体和反相盖玻片上50微升安装媒体上的玻璃显微镜载玻片进行成像。允许幻灯片前成像隔夜干燥。 </li></ol></li><li>收集荧光显微镜图像（根据适当的过滤器设置与所使用的荧光团兼容）通过使用计算机控制的荧光显微镜视频图像捕捉功能。 </li><li>定量荧光信号强度，作为孵化条件的功能。获得的感兴趣区域（ROI）的区域内包围单个小区中的平均像素强度。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+protease+protection+reveals+the+topology+of+the+integral+membrane+protein+Lemur+Tyrosine+Kinase+2+(LMTK2).">Fluorescence protease protection reveals the topology of the integral membrane protein Lemur Tyrosine Kinase 2 (LMTK2).</a> Am J Physiol. 304 (2), C164-C169 (2012).</li><li>Tagawa, A., 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=Assembly+and+trafficking+of+caveolar+domains+in+the+cell:+caveolae+as+stable,+cargo-triggered,+vesicular+transporters.">Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters.</a> J Cell Biol. 170, 769-779 (2005).</li><li>Rizzuto, R., 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=A+gene+specifying+subunit+VIII+of+human+cytochrome+c+oxidase+is+localized+to+chromosome+11+and+is+expressed+in+both+muscle+and+non-muscle+tissues.">A gene specifying subunit VIII of human cytochrome c oxidase is localized to chromosome 11 and is expressed in both muscle and non-muscle tissues.</a> J Biol Chem. 264, 10595-10600 (1989).</li><li>Rizzuto, R., Brini, M., Pizzo, P., Murgia, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+green+fluorescent+protein+as+a+tool+for+visualizing+subcellular+organelles+in+living+cells.">Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells.</a> Curr Biol. 5, 635-642 (1995).</li><li>Lorenz, H., Windl, O., Kretzschmar, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+phenotyping+of+secretory+and+nuclear+prion+proteins+associated+with+inherited+prion+diseases.">Cellular phenotyping of secretory and nuclear prion proteins associated with inherited prion diseases.</a> J Biol Chem. 277, 8508-8516 (2002).</li><li>Ben-Gedalya, T., 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=Cyclosporin-A-induced+prion+protein+aggresomes+are+dynamic+quality-control+cellular+compartments.">Cyclosporin-A-induced prion protein aggresomes are dynamic quality-control cellular compartments.</a> J Cell Sci. 124, 1891-1902 (2011).</li><li>Rogers, M., 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=Epitope+mapping+of+the+Syrian+hamster+prion+protein+utilizing+chimeric+and+mutant+genes+in+a+vaccinia+virus+expression+system.">Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system.</a> J Immunol. 147, 3568-3574 (1991).</li><li>Lisenbee, C. S., Karnik, S. K., Trelease, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+and+mislocalization+of+a+tail-anchored+GFP+redefines+the+identity+of+peroxisomal+ER.">Overexpression and mislocalization of a tail-anchored GFP redefines the identity of peroxisomal ER.</a> Traffic. 4, 491-501 (2003).</li><li>Brandee, A. 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=Mislocalization+and+degradation+of+human+P23H-Rhodopsin-GFP+in+knockin+mouse+model+of+retinitis+pigmentosa.">Mislocalization and degradation of human P23H-Rhodopsin-GFP in knockin mouse model of retinitis pigmentosa.</a> Invest Ophthalmol Vis Sci. 52, 9728-9736 (2011).</li><li>Kuznetsov, A. V., 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=Analysis+of+mitochondrial+function+in+situ+in+permeabilized+muscle+fibers,+tissues+and+cells.">Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells.</a> Nat Protoc. 3, 965-976 (2008).</li></ol>

The authors declare no conflict of interest.

膜蛋白的正确方向和拓扑是为他们的正常功能是必不可少的。尽管认识膜蛋白拓扑的重要性，有许多蛋白质​​的量这样的数据被完全缺乏。 FPP的提供确定膜蛋白拓扑的一个简单而有效的方式，并且是可以由大多数实验室中进行。在FPP方法比以前的方法测定蛋白质拓扑能提供显著优势。例如，没有必要有定向感兴趣31的蛋白质的再次各种域上的多个抗体的面板。在许多情况下，抗体的这?...

质膜，以及众多的细胞内膜，作为分离两个隔室的含水的障碍。在质膜的情况下，该分离是外部之间和小区内;对于细胞内细胞器是细胞质和细胞器内腔之间。例如，内质网（ER）膜分离的ER从胞质还原环境1管腔内氧化环境。膜蛋白合成的雌激素受体 ​​相关的核糖体，并实现其内质网膜2内做出最终的拓扑结构。收购合适的膜方向和拓扑的蛋白质是其正常功能的关键。正确拓扑允许膜蛋白有关的结构域与其结合配偶相互作用，它允许发生临界翻译后修饰，并在质膜蛋白质的情况下，允许细胞进行交互和响应吨o它的环境。充分理解的膜蛋白的功能，它显然是必须知道这样的蛋白质被定向相对于在其中驻留的膜， 也就是说 ，它的膜拓扑结构。除了 ​​获得的基础科学知识，理解的膜蛋白和其中蛋白质表面的各方面暴露于不同环境的拓扑已标记的临床意义，因为膜蛋白包含多数药理靶3。直到最近，接近已确定需要时间和金钱的大量投资或已经所需的试剂，是很难得的膜蛋白的拓扑结构。 既，在硅片的实验和方法已被用于确定驻留在质膜内蛋白的膜拓扑结构。因为基于INDIVI的评价疏水性跨膜结构域的第一个预测双氨基酸3，众多的预测算法，现已在互联网上，而只要求蛋白质的氨基酸序列的知识。然而，假设常常是中央到这样的建模程序，假设，可导致拓扑4,5的不正确的分配。此外，虽然这些基于计算机的预测可以试探性地指派跨膜区域，它们不总是确定的氨基或蛋白质的羧基末端是否在细胞质中，细胞器腔或细胞外。即使具有增加的计算能力，以及利用机器学习算法6中，这样的数据仍然是一个模型，并且必须用实验获得的数据进行验证。直接的实验测定膜拓扑结构采用了与整个蛋白质，其中已经取得了他们的免疫反应的评估之前和细胞通透性后散发称为抗原决定簇的单克隆抗体的面板已经开展。此方法要求一组抗体，其可能不提供对感兴趣的蛋白质。 另一种策略是工程表位的标记，如myc基因或血凝素（HA）到各个位置的蛋白质，再其次是确定免疫前和膜通透性后。除了 ​​免疫标记，酶促标记（包括碱性磷酸酶，β半乳糖苷酶，或β内酰胺酶）和化学修饰，如半胱氨酸扫描都被用来确定膜蛋白拓扑7,8。用于质膜蛋白的拓扑映射的附加方法依赖于一个稍微不同的方式来表位标签。在该方法的标签序列是N联糖基化的共有序列NXS / T。由于糖基化作用，只有当这样的序列是存在于所述生物合成途径中，标签在一管腔相对于存在的内腔发生一个胞质区室是很容易观察到在SDS-PAGE凝胶质量偏移。这样的方法已被应用到multispanning离子通道CFTR 9。虽然所有这些方法都在使用，但很明显，它们都需要在分子生物学相当大的投资，以产生和测序所述歧管结构。 以确定关于位于细胞内细胞器内的跨膜蛋白拓扑信息已被证明是更具挑战性。基于荧光技术的应用然而，已经取得了膜拓扑简单了很多的决心。的双分子荧光互补（附设）的技术依赖于一种荧光蛋白的两个非荧光片段之间的相互作用，恢复该蛋白10的荧光性质。虽然用于确定在体内 10的蛋白质-蛋白质相互作用的最初描述，这种方法也被用于以确定膜蛋白拓扑在植物细胞11。然而，该方法也耗费时间，因为它需要的产生不仅融合蛋白与感兴趣的蛋白质，但也有多种靶向胞质溶胶或细胞器管腔融合蛋白的，并且需要知道哪些细胞内膜蛋白的兴趣在于。 另一种更简单的方法来判断膜蛋白的拓扑结构已被描述12。在测定中，荧光蛋白酶保护（FPP）要求GFP和目的基因之间的融合蛋白的产生。该方法是基于非特异性蛋白酶的GFP部分的相对无障碍;根据是否GFP是由驻留在细胞内的细胞器的管腔免受蛋白水解或通过存在于胞质溶胶暴露于蛋白水解。因此，如果在感兴趣的蛋白质的GFP部分朝向细胞质中，它会被暴露于蛋白酶活性和荧光信号丢失。相反，如果在感兴趣的蛋白质的GFP部分面临来自蛋白酶的环境“保护的”（如高尔基管腔），则荧光信号将持续存在。 以允许蛋白酶进入细胞，但不进入细胞内的膜隔室的胆固醇结合药毛地黄皂苷被使用。胆固醇是在脊椎动物中主要的甾醇，并在质膜相对于细胞内区室是特别丰富。糖苷毒素，毛地黄皂苷，是从植物洋地黄萃取。毛地黄皂苷具有丰富胆固醇的膜具有亲和性，在那里它会导致选择性隔膜permeabiliztion 14,16（ 图1）。除了允许小胞质成分出口，毛地黄皂苷透化还允许外源性分子，例如蛋白酶K或胰蛋白酶的条目。在FPP协议取FAC优势吨的质膜含有高达80％的细胞内胆固醇17的，而其它的细胞器，如ER，高尔基体，内体，线粒体，它们具有非常低的胆固醇含量，保持完整14。胆甾醇的选择性掺入质膜在许多真核细胞中被观察到，允许使用毛地黄皂苷依赖性质膜透化等不同的真核物种如S.酵母对人体14,18。对FPP分析提供了确定（a）一种蛋白质是否是膜结合的/关联的或在胞质溶胶和（b），该膜蛋白的结构域自由扩散朝向胞质溶胶或细胞器内腔的一个简单，快速，相当坚固的装置。应的膜蛋白有多个方向，信号将产生于主要形式和次要形式将不会被检测到。而可以有一些担心，另外一个GFP的部分与上感兴趣的蛋白的可能影响其功能和/或亚细胞定位，这实际上比实际更多的理论。实际上，许多研究已经清楚地表明，将GFP标记不改变蛋白质19,20的属性。

<table border="0" cellpadding="0" cellspacing="0" width="370">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:111px;">Name</td>
			<td style="width:80px;">Company</td>
			<td style="width:116px;">Catalgue Number</td>
			<td style="width:64px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digitonin</td>
			<td>Calbiochem</td>
			<td align="right">300410</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Proteinase K</td>
			<td>Sigma</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Sigma</td>
			<td>T3924</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cav1-GFP</td>
			<td>Addgene</td>
			<td align="right">44433</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pAcGFP-1 Golgi</td>
			<td>Clontech</td>
			<td align="right">632464</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polylysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mounting Media</td>
			<td>Dako</td>
			<td>cS704</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining membrane protein topology using fluorescence protease protection (fpp)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

等离子膜通透性 高效质膜透化是通过使用可溶的荧光蛋白质（ 例如 ，绿色荧光蛋白，红色荧光蛋白）（步骤4）的测定。这些蛋白质，当在细胞中表达，可自由在细胞质中扩散，并且当质膜用毛地黄皂苷（ 图2）被透化都将丢失。荧光信号的完全消失，应该发生在10〜60秒毛地黄皂苷应用。所感兴趣的蛋白与细胞内细胞器相关联的确认是通过注意荧?...

Watch this Scientific Journal Video about Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP at JoVE.com

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

确定膜蛋白拓扑使用荧光蛋白酶保护（FPP）

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

determining-membrane-protein-topology-using-fluorescence-protease

Research

JoVE Journal

Biology

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

17.6K 次观看.  Chicago Medical School. Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP. 

视频: Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

蛋白酶荧光检测试剂盒，用它来确定蛋白酶活性

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

科研

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

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

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

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

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

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

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 ...

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

短杆菌肽为基础的荧光分析法，确定小分子的修改脂质双层属性的潜在

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

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 ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

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 ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.