We thank Barbara Windschiegl for her help in establishing SOPs; Dennis Remme for his work on the NanoCalcFX software and Alina Kollmannsperger, Markus Braner and Milan Gerovac for helpful suggestions on the manuscript. The German-Israeli Project Cooperation (DIP) provided by the DFG and the Federal Ministry of Education and Research to R.T., as well as the Federal Ministry of Economics and Technology (ZIM R&amp;D Project) to R.T. and Nanospot GmbH supported this work.

1.大单层脂质体的制备（的LUV） <ol><li>清洁的圆底烧瓶（10毫升体积）用氮气以除去任何灰尘颗粒。冲洗用乙醇圆底烧瓶中。 注:残余乙醇是可以容忍的，并且不影响随后的步骤。 </li><li>洗1毫升玻璃注射器3倍，氯仿，以消除任何污染。加入4毫升SoyPC20（25毫克/毫升的氯仿溶液）向圆底烧瓶中并用另外DOPE ATTO 390的脂质溶液掺杂到0.1摩尔％的最终浓度。 注意:除了DOPE ATTO 390其他荧光团标记的脂质或亲脂性染料也可使用，这取决于可用的激励源。 </li><li>圆底烧瓶连接到旋转蒸发器。干燥的脂质膜为40分钟，在300毫巴，30℃水浴温度和最大的旋转速度，以在烧瓶的玻璃壁的均匀脂质膜。 </li><li> TRANSF尔烧瓶中在室温下高真空泵和干燥的脂质膜为30分钟。 </li><li>加入5毫升缓冲液（20毫摩尔Tris，150mM的氯化钠，pH值8.0;无菌过滤）和图8的玻璃珠（2.7毫米直径，乙醇洗涤，并干燥）到烧瓶中。将烧瓶连接到旋转蒸发器，并没有在50℃下真空以最大速度旋转。 注意:玻璃珠从玻璃壁机械地位移的脂质膜，导致（多层）囊泡形成。后45分钟旋转的溶液出现乳状和没有残留的脂质膜应该在烧瓶壁可见。代替玻璃珠其他方法，如超声处理该烧瓶在水浴超声波仪，涡旋烧瓶或温和的补液方法也是适用的。 </li><li>在惰性气体（氩气）的烧瓶转移到超声波浴声处理，在室温下4分钟。 注意:超声冲击波扰乱脂质体，使它们的单层。 </li><li>拆分LUV解决方案入1ml等份。 </li><li>通过冷冻在液氮中，LUV溶液进行5次冷冻/解冻循环，随后在一水浴在40℃下解冻。 注意:这导致破裂并彼此单独的脂质体的自发重排，导致尺寸增大。 </li><li>在最后冷冻步骤存储在-80℃下所有样品。 注意:的LUV将稳定至少6个月。 </li></ol> 2.蛋白质重组<ol><li>在冰上解冻1毫升LUV等分。直接重组前，用挤出一个小型挤出机17倍的脂质体通过400纳米的聚碳酸酯过滤膜。 注意:脂质聚集体将被删除，达到最终的均匀尺寸分布。 </li><li>通过加入4％的Triton X-100（V / V），以0.25％的终浓度不稳定的LUV（体积/体积）并在50℃下孵育5分钟。 注意:脂质体在本工序中使用的量取决于纯化MSCLģ&lt;质量SUP&gt; 22℃使用（见步骤2.3）。净化MSCLģ22℃（来自大肠杆菌 ），为在28进行详细描述。 </li><li>拌纯化MSCLģ22℃，洗涤剂饱和脂质体1:20（W / W）比例，并在50°C孵育另外的30分钟。 </li><li>用于洗涤剂除去添加生物珠在Tris缓冲液（20mM的Tris，150mM的氯化钠，pH值8.0;无菌过滤）的蛋白质/脂质体溶液中，用16毫克（湿重）生物珠每微升用于破坏脂质体在清洁剂步骤2.2。 </li><li>在4℃下在旋转板进行洗涤剂除去过夜（16小时）。 注意:溶液的恒定混合确保最佳洗涤剂除去。 </li><li>后在4℃下的洗涤剂除去商店脂酶和用于在同一天的实验。 </li></ol> 3.活动分析<ol><li>在重组蛋白去污剂采取的100微升等分步完成后，2.3不稳定脂蛋白体的解决方案。 </li><li> 1体积的钙黄绿素（30毫摩尔）添加到蛋白 - 脂质体溶液中并在50℃孵育额外的5分钟。 </li><li> 2.6 - 如在步骤2.4中描述从溶液中除去洗涤剂。 </li><li>洗涤剂除去后平衡尺寸排阻柱（2​​0毫升床体积或更多）用Tris缓冲液（3倍床体积）。 注意:脂蛋白体分离可在室温下进行，但在4℃下保持样品不断建议确保分馏后最好蛋白活性。 </li><li>通过将整个等分试样（200微升）的大小排阻柱从游离的钙黄绿素单独脂蛋白体。使混合物浸泡入列，接着从塔通过顶部连续缓冲应用中使用重力流的蛋白脂质体的溶出。观察含有蛋白脂质体作为在洗脱前的离散橙色带的钙黄绿素。收集的500＆＃分数181，L分别。 注:完全分离的脂蛋白体部分免费之后染料钙黄绿素洗脱会。 </li><li>吸移管将80μl每一部分将其移到微孔板荧光读出，并确定通过荧光发射包含含有蛋白脂质体的钙黄绿素的最高量的分数。在含有在490nm激发520nm处检测荧光。使用脂蛋白​​体部分与以下活性测定最高信号。 </li><li>混合20微升含脂蛋白体的级分用980微升Tris缓冲液在1毫升的荧光比色杯中。 </li><li>在测量用分光光度计520纳米（激发波长490）荧光发射。记录1分钟，然后从原液（160毫米）加入25微升MTSET的（[2-（三甲基铵）乙基] methanethiosulfonate）拌匀。继续录制混合过程。总是准备原液直接使用前新鲜。 注意:一旦在缓冲区MTSET水解解决在30分钟内秒和将是非反应性的。 MTSET可以称重并在-20保持为干粉等分℃直至使用。 注意:一旦激活所述MSCLģ22 C通道打开并释放截留钙黄绿素，effluxing的蛋白脂质体和染料的后续脱猝灭时，导致增加的荧光发射导致的钙黄绿素的局部浓度降低。 </li><li>荧光发射再次达到稳定的平台之后，通过加入50微升的Triton X-100的溶解脂质体（4％体积/体积）。 注意:在理想情况下没有进一步的荧光增加可以观察到，在每一个脂质体暗示活性的蛋白质。 </li></ol>使用纳米粒子跟踪的LUV的4粒度分布测试<ol><li>请注意，只有脂质体或脂蛋白体溶液的最小样本是必要的，使用纳米粒子跟踪器尺寸分布分析。稀释5毫克/ m的脂质体溶液升脂质浓度1:5000（V / V）的缓冲液（20mM的Tris，150mM的氯化钠，pH值8.0;无菌过滤）进行分析，以1毫升的最终体积。使用过滤0.2微米的膜，以消除任何悬浮颗粒如灰尘，将扰乱大小分布测量时用于稀释和事先准备LUV和重构所有的缓冲区。 </li><li>洗流室与超纯水。 </li><li>使用表面标记物集中的光束。注入大约300微升稀释脂质体样品，并立即关闭出口阀以停止流动室内部的流动，而不会引入任何气泡。 </li><li>调整对焦和拍照快门/增益设置，以确保样品检测充足的散射信号。 注:20 - 80信号应该在视场中清晰可见。防止过度拥挤（&gt; 80个信号）作为软件将不能够重叠时跟踪单个信号。 </li><li>转到"捕捉​​"SCREEN。调整的温度控制到25℃，并使用该软件控制捕获持续时间90秒。按"录音"开始录制的时间序列。 注意:当一个新窗口中打开的时间序列的完成。 </li><li>保存时间序列在给定的格式（* .AVI）。对于批处理分析过程中实际的原因保存在一个文件夹中的所有文件。 </li><li>打开出口阀，注入另一个300微升到流室。关闭阀门并重复步骤4.3 - 4.6两次。 </li><li>完成之后所有样品运行洗用5ml超纯水流动室。转到"前处理"画面。设置校正参数的温度= 25℃，粘度= 0.91对该协议中使用的Tris缓冲液。 </li><li>打开批处理窗口，并载入记录的时间系列（高级/批处理/设置文件1-3）。按"GO"开始大小分布分析。 注意:分析软件automati卡利跟踪单个颗粒，并计算基于使用在步骤4.8设置的参数的运动行为它们的直径。 注意:得出的尺寸分布分析会自动保存到同一文件夹中的时间序列文件。 </li><li>另外创建一个报告文件汇总结果（导出/创建报告PDF）。 </li></ol> 5.芯片座准备<ol><li>权衡1.0克MDX4医用级弹性体基和0.1g MDX4固化剂到50毫升反应管中。用玻璃棒充分混合两者。 </li><li>把管放入一个干燥器1小时脱气粘合剂。芯片粘合在30分钟内之后使用它，因为胶粘剂会变硬而且太辛苦的工作。 </li><li>在弹性体脱气（步骤5.1）用氯仿彻底清洗客观载玻片芯片粘接前，要消除任何污染。冲洗使用的玻璃注射器5ml氯仿的幻灯片。收集氯仿在位于下方的烧杯中。清洗后让幻灯片干燥。 注意:请在通风橱下这一步。替代氯仿，也可以使用，如丙酮等有机溶剂。 </li><li>沉积粘接材料的少量到使用枪头（为10微升移液器晶体提示）的盖玻璃。请记住，这些芯片必须装配到8室载玻片支架以后上，并且必须相应地放置在幻灯片。 </li><li>小心在从晶片板时用细尖镊子除去一个芯片和粘合剂对玻璃盖的下拉沉积芯片。确保芯片的涂覆侧朝上。 </li><li>轻轻芯片推到盖玻璃用钝的PE镊子的背面。以保证胶水均匀地分配在芯片下方，小心地来回滑动芯片。关键步骤:确保没有气泡被困芯片底下，因为它会干扰光学成像芯片腔以后。还要确保没有粘合材料污染芯片表面。 </li><li>在机柜干燥器变干成品玻璃盖2小时，在65℃。 注意:芯片仍然涂覆有从它们的制造的保护层始发需要被去除。 </li><li>要除去保护层，执行顺序的溶剂清洗步骤。在芯片的固化，预热的水浴中54℃。 </li><li>放置3个烧杯放入水浴中，充满了异丙醇他们两个人，一个充满丙酮。 </li><li>插入芯片护罩玻璃成滑动架，并将其浸入到第一异丙醇填充盘10分钟。之后将其转移到丙酮填充的盘子，其传送用于最终的洗涤步骤的第二异丙醇盘前10分钟，再孵育并孵育另外10分钟。 </li><li>从盘中取出滑动架和小心地用大量的Ultrapur洗辰水，随后芯片用氮气的轻柔气流干燥。 </li><li>起飞从8孔粘性滑动叠片，并把它向后板凳上。小心地拿起盖玻片，并轻轻按压到朝向井芯片粘幻灯片。让我们在使用前一两分钟的成品芯片座休息。 </li></ol> 6.检测制剂注意:纳米孔的芯片粘接到盖玻璃和由8良好粘性滑动分离具有的权益，该多个芯片可以在单一实验运行来解决，与芯片完全被彼此分开。 <ol><li>芯片持有者准备后，用清水冲洗纯乙醇每个芯片和吹干用氮气，以去除如灰尘污染的任何。 </li><li>清洁与空气，氧气或氮气的等离子体芯片表面。取决于等离子体清洗时间周期的类型而变化:1分钟，空气和虮进行氧等离子体处理罗根离子在以80％的功率设置0.3毫巴（RF功率中或高）5分钟。 </li><li>等离子清洗后孵育芯片纯乙醇10-15分钟，以确保腔润湿。 </li><li>为缓冲逐步交换乙醇加入400μl的Tris缓冲液（20毫摩尔Tris，150mM的氯化钠，pH值8.0;无菌过滤），接着而不引入气泡并随后除去400微升的溶液混合。重复此过程12次，以保证乙醇去除。 注:乙醇将是有害的脂质体和悬浮脂质双层。 </li><li>纺丝原液的Tris缓冲液（20mM的Tris，150mM的氯化钠，pH值8.0;无菌过滤）用5mM 氯化钙和1μM的Oy647染料。完全从芯片去除洗涤缓冲液，并立即在掺杂缓冲器添加到芯片上。注意:芯片将用洗涤缓冲液除去后的薄缓冲膜覆盖，因此芯片不与空气直接接触，确保了​​腔的恒定润湿和芯片表面。 </li><li>加入1毫克/毫升的LUV或PROTEO-的LUV在Tris缓冲液（20mM的Tris，150mM的氯化钠，pH值8.0;无菌过滤）到每个孔中。 注意:在每个孔中的最终体积为300微升。考虑掺杂Tris缓冲液加入过程中控制染料和化学改性剂MTSET随后添加。 </li><li>用于在芯片表面上的孔隙跨越脂质双层形成孵育芯片在室温下与该脂质体溶液1小时。随后立即用4X 400微升钙 2+ Tris缓冲的每个芯片。 </li><li>控制染料添加到每个孔中，以5微米的终浓度。这种芯片现在准备好了易位检测。 </li></ol> 7.实验设置<ol><li>安装芯片持有者的落射荧光显微镜（20X空中目标，长工作距离）。专注于纳米孔芯片的边缘更容易找到微腔的焦平面。一旦空腔处于焦点扫描纳米孔阵列，用于一个区域在显示器最高密封比例。 注意:每个倒置落射荧光显微镜可以被用于执行芯片测定。根据目的的倍率（20X - 60X）测定腔的数量将有所不同，在使用20X放大率视图中的单个字段高达9000空腔。具有长工作距离的空气的目标是优选的。使用倒置共聚焦激光扫描显微镜（CLSM）也是可能的，但是图像获取可能是限制因素，由于聚焦的有限深度。 </li><li>优化芯片的照明。 注意:确保两个染料通道不超过最大灰度值，作为变化不能被观察到。出口实验调整易位染料的照度的最大信号容量的90％。调整开放式腔体控制染料的最大信号容量的80-90％，清楚地探测到任何泄漏膜密封。 </li><li>一旦所有的通道被调整开始的时间系列。拍摄图像每10-20 SEc代表90分钟。 注:这是足以同样遵循特定的非特定外流事件。 </li><li>初始化通过加入半胱氨酸特异性的荧光底物的流出，带正电荷的化合物MTSET至3毫米的最终浓度。允许至少5分钟MTSET除了前实验运行的，以便能够稍后建立稳定的荧光信号到通道开口基线前。永远新鲜，直接使用之前准备MTSET解决方案。 </li></ol> 8.数据分析<ol><li>与ImageJ的一个标准版本（文件/导入/图像序列）打开时间序列图像堆栈。 </li><li>如果荧光通道堆放，拆个别栈通道每通道（图片/栈/协议栈图像）。 </li><li>重复易位衬底通道中的投资回报率的第一图像（感兴趣的区域）的识别（图像/复制）。 </li><li>将图像转换为二进制图像（图像/调整/阈值）。确保所使用的算法描述了所有密封腔信号不重叠模糊了。 </li><li>自动定义的与粒子分析器工具感兴趣区域（分析/粒子分析）。定义所定义的最小粒径，以避免粒子的噪声。检查选项"排除在边缘"和"有漏洞"。确保投资回报结果反映微阵列模式。中间的投资回报率是文物，因此将其删除。保存ROI列表。 注意:测量参数已被设置为"区"颗粒分析（分析/设定测量/面积）之前</li><li>打开时间序列分析仪插件（http://rsb.info.nih.gov/ij/plugins/time-series.html），它会自动调整所有ROI大小相同。重新大小的所有现有的投资回报为6×6像素和椭圆形（定时器系列分析仪/ AutoROIProperties）的宽度/高度。选中选项"调整现有ROIS"。保存生成的调整ROI名单。 </li><li>打开ROI经理（分析/工具/ ROI管理器）和加载调整投资回报率清单的时间序列的每个通道（ROI经理/其它/打开）。投资回报率将被叠加。分析各ROI信号变化启动Multi测量刀具（ROI经理/其它/多测量），选择"测量所有切片"和"每片一行"，并启动多措施。 注:测量参数，必须设置这一步，以"平均灰度值"（分析/设置测量/平均灰度值） </li><li>保存结果表，使平均灰度值的* .txt或* .xls格式每个投资回报率。 </li><li>通过"导入文件"选项，导入每个成像通道进入NanoCalcFX的投资回报率分析。设置"使用第一行命名系列"。根据时间序列的图像采集周期设定图像之间的步长大小，并选择相应的列和小数分隔。 </li><li>使用"多片"选项个别萤光灯的图形自动关联rescent通道。 注意:现在的事件可以被分析和使用由3色的光谱信息提供的信息进行分类。 </li><li>适合使用内置的拟合函数（调整选项）选定曲线。 注:实施流出和流入方程适用于所有单指数扩散驱动的事件和合适的边界可以设置。造成速率常数可以使用直方图工具绘制或导出数据的进一步细化。 </li></ol>

<ol>
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The authors declare no competing financial interests.

这里提出的技术允许膜蛋白转运的一个高度并行分析。重构膜蛋白系统可以直接应用到生物芯片，使得理论上每膜转运的适应或信道成为可能。运输分析仅由建立的荧光读出系统的任通过直接荧光变化（荧光团的易位或荧光标记的底物）或间接荧光变化（pH敏感染料，次级酶反应）的限制。后者，但是还没有建立。 上的单个蛋白质水平膜通道或转运的功能特性是该技术的主要...

膜蛋白质的分析已经成为过去20年中增加了对基础和制药研究兴趣。新型药物的开发依赖于识别和新的目标细致的刻画，目前正在限制因素之一。即所有的药物靶标-约60％是膜蛋白1的事实，使得技术的发展，以阐明其功能最重要的。 在过去，电的通道和转运的研究技术已经开发在众多2 - 4。在相反的非电的基材呈现出更为艰巨的任务。然而，它们特别感兴趣作为首要的药物靶标，因为它们控制穿过细胞膜和功能键受体溶质和营养物质的熔剂在信号级联5。 相当大的努力已投入T的发展echniques研究膜转运蛋白6,7的功能。 10，包括固体支持的脂质双层，拴系双层11,12 microblack脂膜13，14和原生泡囊阵列15,16仅举几-使用固体支持膜系统已在本领域8成为最有希望的工具。他们中的一些甚至可以作为商业设置17，18。一些例子已经发表结合研究单膜蛋白以高度平行的方式14，19，为筛选应用的先决条件的能力。然而，这些方法很少从基础研究到工业环境弥合。的困难往往在于该系统是自动化的能力，成本密集的生产和/或费力的制剂。一种方法Òvercoming上述所有障碍是最终目的。 这里介绍的技术的开发，研究膜通道和载体在体外对单个蛋白质水平20受控的环境- 22。 26或黑脂膜27 -净化膜蛋白的重建成的LUV是远远超过了GUVs 23类似的方法建立的。它们可以直接施加到芯片表面，其中双层形式经由自组装过程的发生。纳米多孔芯片（ 图 1）的玻璃底设计允许空气显微镜，其允许系统的简单的自动化。在用电动载物台相结合的多个芯片可以在同一时间进行测定，用包含数千个密封腔的用于分析每个视野。 图 1。复纳米孔生物芯片 。A） 设计硅绝缘体（SOI）晶片由反应离子刻蚀结构。大约1150个人的芯片是由具有相同属性和质量。B）每个芯片包含250,000纳米孔径个人微腔每片晶圆制造。比例尺:200微米C）每个腔是通过多光谱荧光读出寻址。一个不透明的顶层块从缓冲储荧光信号，使得生物芯片倒置荧光显微镜。D）的原子力显微镜兼容（AFM）成像揭示均匀排列孔开口和3.6纳米的氧化硅层的表面粗糙度（N = 40）最优的囊泡融合。比例尺:5微米E）扫描电子麦克风roscopy（SEM）图像示出了通过纳米孔允许访问在硅芯片内的飞升腔的横截面。这个数字是从21重复使用许可。 <a href="https://www.jove.com/files/ftp_upload/53373/53373fig1large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> 使用免费软件，以保证最终用户不受限制的访问进行所有的数据分析。时间序列使用免费的图像处理软件和一个自定义生成曲线分析软件，使批量处理和曲线的多种荧光渠道和数千家大型数据集的简单分析相关性。 在这个协议中使用的模型蛋白是大电导（元富）通道蛋白从大肠杆菌产生的机械敏感性通道大肠杆菌 。它用作一个阀以释放在自然界渗透压休克，但在设计合理SYNT这样一种方式被修改hetic官能可共价地附连到信道收缩侧。通过信道被触发以打开共价结合活化剂（MTSET）的电荷斥力，产生的纳米阀。像离子，水，小分子蛋白，但也小的荧光小分子可以通过渠道渗透。这里，该蛋白质作为模型来演示系统来检测蛋白介导的转运的能力。

<table border="0" cellpadding="0" cellspacing="0" width="1093">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Reagent</td>
			<td style="width:232px;"></td>
			<td style="width:305px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">[2-(Trimethylammonium)ethyl] 
			methanethiosulfonate</td>
			<td style="width:232px;">Toronto Research Chemicals Inc.</td>
			<td style="width:305px;">T792900</td>
			<td>MTSET; hydrolized by water. Keep as dry pouder aliquot at -80 &#176;C. Use immediately (30 minutes) after solubilization in buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">1 ml gas-tight syringe</td>
			<td style="width:232px;">Hamilton</td>
			<td style="width:305px;">#1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">10 ml round flask</td>
			<td style="width:232px;">Schott Duran</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">2.7 mm glas beads</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">N032.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">2-Propanole</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">9866.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">30 cm Luer-Lock Extension Tube</td>
			<td style="width:232px;">Sarstedt</td>
			<td style="width:305px;">744304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Acetone</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">5025.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Bio-Beads SM-2 Adsorbent</td>
			<td style="width:232px;">Bio-Rad</td>
			<td style="width:305px;">152-3920</td>
			<td>need to be activated before first use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:389px;">CaCl2 Dihydrat</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">HN04.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Calcein</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:305px;">C0875</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Chloroform reagent grade</td>
			<td style="width:232px;">VWR Chemicals</td>
			<td style="width:305px;">22711324</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:389px;">DOPEATTO390</td>
			<td style="width:232px;">ATTO-TEC</td>
			<td style="width:305px;">AD 390-165</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Ethanol absolute</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:305px;">32205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Injekt Single-use syringe</td>
			<td style="width:232px;">Braun</td>
			<td style="width:305px;">460 60 51V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Injekt-F single-use syringe</td>
			<td style="width:232px;">Braun</td>
			<td style="width:305px;">91 66 017V</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:389px;">Keck clips</td>
			<td style="width:232px;">Schott</td>
			<td style="width:305px;">KC29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">L-&#945;-Phosphatidylcholine, 20% (Soy)</td>
			<td style="width:232px;">Avanti Polar Lipids</td>
			<td style="width:305px;">5416016</td>
			<td>store under inert gas at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NaCl 99.5% p.a.</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">3957.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nanopore E100 wafer/chips</td>
			<td style="width:232px;">Micromotive (Mainz/Germany)</td>
			<td style="width:305px;"></td>
			<td>available on request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nucleopore Track-Etch Membrane 0.4 &#181;m</td>
			<td style="width:232px;">Whatman</td>
			<td style="width:305px;">800282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Oregon Green Dextran 488 (70 kDa)</td>
			<td style="width:232px;">life Technologies</td>
			<td style="width:305px;">D-7173</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Oy647</td>
			<td style="width:232px;">Luminartis (M&#252;nster/Germany)</td>
			<td style="width:305px;">OY-647-T-1mg</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Rotilabo-syringe filtration, unsterile, pore-size 0.22 &#181;m</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">P 818.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Sephadex G-50</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:305px;">G5080</td>
			<td>column material for size exclusion chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Silastic MDX4-4210</td>
			<td style="width:232px;">Dow Corning</td>
			<td style="width:305px;"></td>
			<td>curing agent for chip fixation onto cover glass support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">sticky-Slide 8-well</td>
			<td style="width:232px;">ibidi</td>
			<td style="width:305px;">80828</td>
			<td>multi-well chamber for the mounting onto glass slides (chip holder)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Three-way stopcock blue</td>
			<td style="width:232px;">Sarstedt</td>
			<td style="width:305px;">744410001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Tris Pufferan 99.9% Ultra Quality</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">5429.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Triton-X 100</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">6683.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Whatman 0.2 &#181;m cellulose nitrate membrane filter</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">NH69.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Name</td>
			<td style="width:232px;">Company</td>
			<td style="width:305px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Equipment</td>
			<td style="width:232px;"></td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">B&#252;chi 461 water bath</td>
			<td style="width:232px;">B&#252;chi</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">B&#252;chi Rotavapor RE 111</td>
			<td style="width:232px;">B&#252;chi</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Cary Eclipse Fluorescence Spectrophtometer&#160;</td>
			<td style="width:232px;">Varian</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">LiposoFast Mini Extruder</td>
			<td style="width:232px;">Avestin</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Membrane pump&#160;</td>
			<td style="width:232px;">Vaccubrand</td>
			<td style="width:305px;">15430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nanosight Nanoparticle Tracking Microscope</td>
			<td style="width:232px;">Malvern / Nanosight</td>
			<td style="width:305px;">LM 14C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NyONE microscope</td>
			<td style="width:232px;">Synentec</td>
			<td style="width:305px;"></td>
			<td>available on request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Pump control</td>
			<td style="width:232px;">Vaccubrand</td>
			<td style="width:305px;">CVC 2II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Sonicator bath Sonorex RK100H</td>
			<td style="width:232px;">Brandelin electronic</td>
			<td style="width:305px;">31200001107477</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Vaccum pump RC5</td>
			<td style="width:232px;">Vaccubrand</td>
			<td style="width:305px;">1805400204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Water bath W13</td>
			<td style="width:232px;">Haake</td>
			<td style="width:305px;">002-9910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Plasma Cleaner PDC-37G</td>
			<td style="width:232px;">Harrick Plasma</td>
			<td style="width:305px;">PDC-37G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Name</td>
			<td style="width:232px;">Company</td>
			<td style="width:305px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Software</td>
			<td style="width:232px;"></td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">ImageJ</td>
			<td style="width:232px;">Open Source</td>
			<td style="width:305px;">http://imagej.nih.gov/ij/</td>
			<td>scientific image processing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NanoCalcFX</td>
			<td style="width:232px;">Freeware</td>
			<td style="width:305px;">http://sourceforge.net/projects/nanocalc/</td>
			<td>data analysis/evaluation software for massive transport kinetic datasets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NTA 2.3 Analytical Software</td>
			<td style="width:232px;">Nanosight</td>
			<td style="width:305px;"></td>
			<td>data acquisition and analysis software for nanoparticle tracking microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NTA 2.3 Temperature Comms</td>
			<td style="width:232px;">Nanosight</td>
			<td style="width:305px;"></td>
			<td>temperature controle software for nanoparticle tracking microscope</td>
		</tr>
	</tbody>
</table>

membrane transport processes analyzed by a highly parallel nanopore chip system at single protein resolution

上的单个蛋白质水平的膜蛋白运输仍然逃避详细的分析，如果易位衬底是非电的。相当大的努力在这一领域已经取得，但技术实现自动化高通量运输分析与膜转运的分析所需的无溶剂型脂质双层技术组合是罕​​见的。然而，这类转运体是细胞动态平衡至关重要的，因此迫切需要在药物开发和方法，以获得新的见解的主要目标。 在这里介绍稿件描述了一种新的生物芯片的膜蛋白介导的运输过程中的单一转运分辨率分析建立和处理。生物芯片是由通过纳米孔是在它的设计高度并行，可在工业级和数量来制造封闭微腔。蛋白窝藏脂质体可直接被应用到在芯片表面上形成使用SSM的技术自组装孔跨越脂质双层（固体支持的脂质膜）。孔横跨膜的部件是独立的，为基片易位接口进入或离开腔室空间，它可以随后在实时多光谱的荧光读数。标准操作规程（SOP）建立允许简单的建立几乎每一个膜蛋白的芯片表面，可以进行功能上的重组蛋白窝藏脂质双层的。唯一的先决条件是对非电的输送基板建立了荧光读出系统。 高内涵筛选的应用是通过使用自动化倒置荧光显微镜并行记录多个芯片的accomplishable。可以使用免费的定制设计分析软件进行分析大型数据集。三色多光谱荧光读出还允许无偏数据判别成不同的事件类，消除假阳性结果。 芯片技术目前基于SiO 2的表面上，但使用涂覆金的芯片表面进一步官能也是可能的。

孔横跨膜可以很容易地在一个自组装方式的纳米结构化芯片表面上产生。然而，潜在的过程仍然细腻许多参数，如脂质体大小，脂质体人口，层数，脂质和盐的浓度和化学表面特性的单分散性的影响。大多数这些参数已经仔细表征和在上述协议标准化。其他参数应但是每一个新的准备过程中进行检查，以确保成功的实验。这些脂质体大小分布和人口分散度（图</strong...

Watch this Scientific Journal Video about Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution at JoVE.com

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

膜运输过程由单一的蛋白质解析一个高度并行的纳米孔芯片分析系统

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

membrane-transport-processes-analyzed-highly-parallel-nanopore-chip

Research

JoVE Journal

Biology

11.6K 次观看.  Goethe University Frankfurt. The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

视频: Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

科研

生物学

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

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

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

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

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

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

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 ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

在大气压力质谱直接分析单细胞

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

视黄醇运输的血浆视黄醇结合蛋白的膜受体的实时分析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

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 ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

方法来研究上皮转运蛋白功能和表达在纯肠和Caco-2细胞生长在3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...