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>

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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焦点。在使用自动显微镜组合，蛋白质效应建立的筛选应用也是可能的。该测定法设置允许的最多8个芯片的并行记录，与每个芯片上的多个检测点的。因为芯片的完全分离的，它是真正的平行，允许多个实验重复包括控制在一个单一实验运行，一旦条件已经成立。 设置实验中的关键步骤是真正单层囊泡的制备中，所选择的蛋白质的活性重组，芯片表面和悬浮脂质双层朝向在测定中使用的基板的保持能力上扩频脂蛋白体的效率。 而单层囊泡的制剂的基本重要性该测定，以避免建立悬浮脂质双层形成期间在芯片表面上的多层膜薄片，它是相当容易的通过在制备过程中超声处理的脂质体，以确保囊泡unilamellarity。 蛋白脂质体的直接应用是方法presente的一大进步ð。相较于以前的方法更复杂和医学相关的蛋白质，现在可以有针对性的，需要当然事先建立一个重建协议，得到所选择的功能性蛋白中的LUV的。此外，拥有大型膜外域的膜蛋白减少正确的SSM的形成。这必须通过改变的CaCl 2浓度为每一个新的蛋白质进行了优化，例如。 目的蛋白质的重组比例，必须慎重选择。单个蛋白事​​件的分析期望和accomplishable使用这里提出的技术。因此重新构成的蛋白质的量应不超过每孔横跨膜的膜片假设的理想重建和膜蛋白跨双层随机分布1-2功能性蛋白质的单位。此外重构后的蛋白质的方向性还应当牢记，因为它有n个OT既方向性之间的平等。 最后，一​​旦所有这些问题都仔细检查和优化，没有使用的基板可以显示孔跨越膜的非特异性任何膜通透性。带正电的荧光，犹如红色光谱政权大多数基于罗丹明染料往往会迅速克服膜屏障。强疏水性相互作用也可导致非特异性附着于脂质双层。这两个属性是不希望的。 离子通道的一个定性的方式研究也可想而知。筛选使用二进制是渠道效应/无读数可能会想到用离子敏感染料。这里的最大障碍是建立一个至少部分离子保留脂质双层。这可能仅仅是通过纳米孔芯片的表面改性实现的。

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.remotexs.ntu.edu.sg/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><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>[2-(Trimethylammonium)ethyl]&lt;br/&gt; methanethiosulfonate</td><td>Toronto Research Chemicals Inc.</td><td>T792900</td><td>MTSET; hydrolized by water. Keep as dry pouder aliquot at -80 &amp;deg;C. Use immediately (30 minutes) after solubilization in buffer.</td></tr><tr><td>1 ml gas-tight syringe</td><td>Hamilton</td><td>#1001</td><td></td></tr><tr><td>10 ml round flask</td><td>Schott Duran</td><td></td><td></td></tr><tr><td>2.7 mm glas beads</td><td>Roth</td><td>N032.1</td><td></td></tr><tr><td>2-Propanole</td><td>Roth</td><td>9866.5</td><td></td></tr><tr><td>30 cm Luer-Lock Extension Tube</td><td>Sarstedt</td><td>744304</td><td></td></tr><tr><td>Acetone</td><td>Roth</td><td>5025.5</td><td></td></tr><tr><td>Bio-Beads SM-2 Adsorbent</td><td>Bio-Rad</td><td>152-3920</td><td>need to be activated before first use</td></tr><tr><td>CaCl&lt;sub&gt;2 &lt;/sub&gt;Dihydrat</td><td>Roth</td><td>HN04.3</td><td></td></tr><tr><td>Calcein</td><td>Sigma</td><td>C0875</td><td>store dark at -20 &amp;deg;C</td></tr><tr><td>Chloroform reagent grade</td><td>VWR Chemicals</td><td>22711324</td><td></td></tr><tr><td>DOPE&lt;sup&gt;ATTO390&lt;/sup&gt;</td><td>ATTO-TEC</td><td>AD 390-165</td><td>store dark at -20 &amp;deg;C</td></tr><tr><td>Ethanol absolute</td><td>Sigma-Aldrich</td><td>32205</td><td></td></tr><tr><td>Injekt Single-use syringe</td><td>Braun</td><td>460 60 51V</td><td></td></tr><tr><td>Injekt-F single-use syringe</td><td>Braun</td><td>91 66 017V</td><td></td></tr><tr><td>Keck clips</td><td>Schott</td><td>KC29</td><td></td></tr><tr><td>L-&amp;alpha;-Phosphatidylcholine, 20% (Soy)</td><td>Avanti Polar Lipids</td><td>5416016</td><td>store under inert gas at -20 &amp;deg;C</td></tr><tr><td>NaCl 99.5% p.a.</td><td>Roth</td><td>3957.2</td><td></td></tr><tr><td>Nanopore E100 wafer/chips</td><td>Micromotive (Mainz/Germany)</td><td></td><td>available on request</td></tr><tr><td>Nucleopore Track-Etch Membrane 0.4 &amp;micro;m</td><td>Whatman</td><td>800282</td><td></td></tr><tr><td>Oregon Green Dextran 488 (70 kDa)</td><td>life Technologies</td><td>D-7173</td><td>store dark at -20 &amp;deg;C</td></tr><tr><td>Oy647</td><td>Luminartis (M&amp;uuml;nster/Germany)</td><td>OY-647-T-1mg</td><td>store dark at -20 &amp;deg;C</td></tr><tr><td>Rotilabo-syringe filtration, unsterile, pore-size 0.22 &amp;micro;m</td><td>Roth</td><td>P 818.1</td><td></td></tr><tr><td>Sephadex G-50</td><td>Sigma-Aldrich</td><td>G5080</td><td>column material for size exclusion chromatography</td></tr><tr><td>Silastic MDX4-4210</td><td>Dow Corning</td><td></td><td>curing agent for chip fixation onto cover glass support</td></tr><tr><td>sticky-Slide 8-well</td><td>ibidi</td><td>80828</td><td>multi-well chamber for the mounting onto glass slides (chip holder)</td></tr><tr><td>Three-way stopcock blue</td><td>Sarstedt</td><td>744410001</td><td></td></tr><tr><td>Tris Pufferan 99.9% Ultra Quality</td><td>Roth</td><td>5429.2</td><td></td></tr><tr><td>Triton-X 100</td><td>Roth</td><td>6683.1</td><td></td></tr><tr><td>Whatman 0.2 &amp;micro;m cellulose nitrate membrane filter</td><td>Roth</td><td>NH69.1</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B&amp;uuml;chi 461 water bath</td><td>B&amp;uuml;chi</td><td></td><td></td></tr><tr><td>B&amp;uuml;chi Rotavapor RE 111</td><td>B&amp;uuml;chi</td><td></td><td></td></tr><tr><td>Cary Eclipse Fluorescence Spectrophtometer&amp;nbsp;</td><td>Varian</td><td></td><td></td></tr><tr><td>LiposoFast Mini Extruder</td><td>Avestin</td><td></td><td></td></tr><tr><td>Membrane pump&amp;nbsp;</td><td>Vaccubrand</td><td>15430</td><td></td></tr><tr><td>Nanosight Nanoparticle Tracking Microscope</td><td>Malvern / Nanosight</td><td>LM 14C</td><td></td></tr><tr><td>NyONE microscope</td><td>Synentec</td><td></td><td>available on request</td></tr><tr><td>Pump control</td><td>Vaccubrand</td><td>CVC 2II</td><td></td></tr><tr><td>Sonicator bath Sonorex RK100H</td><td>Brandelin electronic</td><td>31200001107477</td><td></td></tr><tr><td>Vaccum pump RC5</td><td>Vaccubrand</td><td>1805400204</td><td></td></tr><tr><td>Water bath W13</td><td>Haake</td><td>002-9910</td><td></td></tr><tr><td>Plasma Cleaner PDC-37G</td><td>Harrick Plasma</td><td>PDC-37G</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td>Open Source</td><td>http://imagej.nih.gov/ij/</td><td>scientific image processing software</td></tr><tr><td>NanoCalcFX</td><td>Freeware</td><td>http://sourceforge.net/projects/nanocalc/</td><td>data analysis/evaluation software for massive transport kinetic datasets</td></tr><tr><td>NTA 2.3 Analytical Software</td><td>Nanosight</td><td></td><td>data acquisition and analysis software for nanoparticle tracking microscope</td></tr><tr><td>NTA 2.3 Temperature Comms</td><td>Nanosight</td><td></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 2 的A）。为了达到最佳的孔隙跨越膜的形成和避免脂质体侵入到空腔空间脂质体大小以及细孔径以上是必要的。此外，单分散脂质体制剂已被证明，得到在膜最好的结果扩展和副食上。具有要检查的每一个制备的另一个关键步骤是复原后的蛋白质的活性。使用用于与MSCLģ22℃重构脂质体荧光脱猝灭测定中，通道开口可通过荧光光谱进行监控，从而脂质体窝藏活性MSCLģ22℃蛋白（图 2 B）的比率。 一旦扩散到芯片表面，在配体活化的荧光靶的溶质易位MSCLģ22℃可以遵循（图 3 的B）。工程MSCL 摹 22℃通道关闭其默认状态，包埋腔空间内小荧光分子。通过加入化学调制器MTSET的正电荷被引入到的收缩区域在MSCL 摹 22℃孔，推动它通过开放的静电斥力。现有包埋荧光逃避腔空间，实现扩散均衡。荧光的型腔空间内的后续的降低可以被监视。该方法是高度可再现的，从而导致外排事件的均匀群体（ 图 3℃）。将所得流出曲线簇在两个不同的群体，这表明该测定的单和多通道易位事件（ 图 3 D）之间进行区分的能力。此外，该系统能够跟踪多达三个光谱以及分离染料在并行和实时地，使得能够记录高含量掩护和之间阳性，假阳性和阴性结果精确，客观地辨别。在这项研究中9.046密封腔进行了分析，其中的8％显示EFFLUX行为。部署不同的荧光读出的信道，事件可以被识别成单指数流出事件，复杂动力学，脂质侵扰和膜破裂（图 3 E）。只有显示控制溶质和膜染料稳定的信号事件被认为是最后的分析。复杂的动力学表示具有非恒定的控制信号外排事件，因此被省略。脂质入侵和膜破裂是用孔跨越脂质双层系统本质上发生的神器事件。它们可以在控制染料信号之上（ 图 4）如所述被丢弃。 <img alt="图2" src="/files/ftp_upload/53373/53373fig2.jpg" /> 图 2. 大小脂酶和MSCL功能分布 。A）囊泡analyz<html>由纳米粒子跟踪分析主编。 LUV样品前（黑色曲线）和MSCL 摹 22 C（红色曲线）的重组后进行了检查。 LUV制备后的214±70nm的平均粒径达到。大小在大小。B）中的MsCl活动158±60纳米的脂蛋白体重建期间减少，导致用一个流出测定复原后的验证。28自猝灭钙黄绿素从脂蛋白通过MSCL信道的MTSET介导的开口释放导致荧光团（红）和荧光强度的快速增加的脱猝灭。没有蛋白质的脂质体显示没有这样流出，即使经过多次MTSET加成（黑色）。脂质体释放的后续洗涤剂溶解介绍（TX​​-100），所有以前封装的染料。这一数字与21许可复用。等="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53373/53373fig3.jpg" /> 图 3。在单分子水平由元富配体门控溶质易位 。A）工程MSCL 摹 22℃通道在其默认状态关闭。小分子溶质如荧光团都无法通过通道也不脂质双层通过两者都不是。在加入MTSET，各加入MsClģ22℃原聚体的工程化单半胱氨酸被修改，引入在五聚体通道复合物的收缩侧的多个正电荷。所述MSCLģ22 C通道通过电荷排斥推开并且包裹的染料能够扩散出空腔，从而导致降低的荧光强度。B）中钛我跟踪MSCL 摹 22℃开在加入MTSET的。包含功能再造MSCL 摹 22℃脂蛋白铺展在芯片表面。 Oy647（红色）被截留空腔作为易位基板内部。 OregonGreen葡聚糖（70 kDa的，绿色）作为信道不可渗透控制基板监测在实验过程中传输事件的底物特异性以及膜的完整性。分别的LUV辅以DOPE ATTO 390（0.1％（摩尔）;紫色）以检查内部腔体脂肪污染。在t = 0秒MTSET的增加（3毫米最终）打开通道，导致从空腔Oy647流出，通过荧光读出。c中记录）随机挑选MSCL 摹 22℃流出曲线表现出典型的外流事件。D）率易位事件常数（n = 242）簇分成两个高斯人口S，这表明单和多通道传输事件可以被识别。E）高内涵筛选和三色实时读出的9046片密封腔统计分析。所有分析的芯片腔的8％，显示指数外流事件。由于频谱解码（红色:易位溶质;绿色:控制溶质;紫:脂肪），外流事件，复杂的动力学，脂质入侵和膜破裂可能会受到歧视，从而消除误报。这个数字是从21重复使用许可。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53373/53373fig3large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53373/53373fig4.jpg" /> 图 4。事件类别由三色检测区分 。易位溶质（红），CONTRO升溶质（绿色）和脂质染料（紫色），可以光谱判别允许无偏的数据选择。A）中密封腔携带无膜蛋白显示了MSCL通道记录所述的实验条件下，无焊剂的事件（参照图3A） 。荧光读数是所有通道稳定。B）不明确暂停脂双层能够闯入在不连续的脂双层腔。C）膜破裂导致跨越纳米孔裂缝。由此，腔体空间不再从缓冲室分离。包埋染料（红色）回避腔下来浓度梯度。控制溶质（绿色），先前无法穿过膜传递，进入对脂质入侵事件的空腔。D）的实施例。而对于脂质染料（紫色）的荧光信号由于膜侵入空腔空间增大，易位溶质是仆射D OUT空腔（红色），导致荧光信号的减少。纳米孔是由脂双层阻挡，防止控制基板（绿色）。E）的通过期间膜破裂，封闭的运输溶质迅速地从所述腔（红色）逃避，而控制溶质中（绿色扩散）。这个数字是从21重复使用许可。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53373/53373fig4large.jpg" target="_blank">请点击此处查看本图的放大版本。</a>

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

Nanyang Technological University

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

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

科研

生物化学

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the creation of a nanopore in a thin insulating membrane remains challenging. Fabrication methods involving specialized focused electron beam systems can produce well-defined nanopores, but yield of reliable and low-noise nanopores in commercially available membranes remains low2,3 and size control is nontrivial4,5. Here, the application of high electric fields to fine-tune the size of the nanopore while ensuring optimal low-noise performance is demonstrated. These short pulses of high electric field are used to produce a pristine electrical signal and allow for enlarging of nanopores with subnanometer precision upon prolonged exposure. This method is performed in situ in an aqueous environment using standard laboratory equipment, improving the yield and reproducibility of solid-state nanopore fabrication.

A methodology for preparing solid-state nanopores in solution for biomolecular translocation experiments is presented. By applying short pulses of high electric fields, the nanopore diameter can be controllably enlarged with subnanometer precision and its electrical noise characteristics significantly improved. This procedure is performed in situ using standard laboratory equipment under experimental conditions.

微调尺寸和最小化固态纳米孔的噪音

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the ...

工程学

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

监测与固态纳米孔蛋白质吸附

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

生物工程

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

单金属纳米粒子的高分辨率物理特性

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

化学

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in nanoscience. However, electrochemically analyzing single nanoparticles is challenging, as the sensing nanointerface is uncontrollable. To address this challenge, we describe here the fabrication and characterization of a closed-type wireless nanopore electrode (WNE) that exhibits a highly controllable morphology and outstanding reproducibility. The facile fabrication of WNE enables the preparation of well-defined nanoelectrodes in a general chemistry laboratory without the use of a clean room and expensive equipment. One application of a 30 nm closed-type WNE in analysis of single gold nanoparticles in the mixture is also highlighted, which shows a high current resolution of 0.6 pA and high temporal resolution of 0.01 ms. Accompanied by their excellent morphology and small diameters, more potential applications of closed-type WNEs can be expanded from nanoparticle characterization to single molecule/ion detection and single-cell probing.

Here, we present a protocol for fabrication of a closed-type wireless nanopore electrode and subsequent electrochemical measurement of single nanoparticle collisions.

一种用于分析单纳米粒子的闭式无线纳米粒子电极

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

质量敏感颗粒跟踪，用于表征膜相关的高分子动力学

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...