Research at the Lewinson lab is supported by grants from the Israel Academy of Science, the Rappaport Institute for Biomedical research, the Meriuex Research Foundation, and the Marie Curie career reintegration grant (OL). EV is supported by the Fine scholarship and by the Technion Faculty of Medicine. NLL is supported by grants from the Israel Academy of Science.

1.蛋白质样品和缓冲液准备<ol><li>样品制备:纯化目的蛋白，并确保所有聚集体被除去，通过注入实验前的蛋白上的凝胶过滤柱或通过超速离心（通常为10分钟，在100000×g离心就足够了）。 注:虽然可取，高纯度蛋白制剂不是必须的。 SPR已成功地用于与低于完美纯化，甚至与总提取物8,9。 </li><li>缓冲液配制: <ol><li>制备实验的缓冲液（称为"运行缓冲液"，或包）。对于这里描述的协议中，使用的50mM的Tris-HCl pH为7.5，150mM的NaCl和0.05％（重量/体积）DDM（正十二烷基β-D-麦芽糖苷）。请记住，缓冲组合物的选择可根据所研究的系统可以容易地进行修改。 </li><li>过滤器全部采用0.22微米的过滤器缓冲区和蛋白质样品。确保提前的过滤器是蛋白质兼容。在一般情况下，最近市场上销售的SPR平台包含一个在线脱放气;如果不是这种情况下，解气体之前的缓冲器来使用。 </li><li>夜间透析或稀释样品对RB避免缓冲区不匹配。使用高粘度的化学物质时，透析（或缓冲液交换的任何其他方法）是特别可取的（ 例如 ，蔗糖，甘油，DMSO）。之前对RB瓶连接至泵入口保持预留的10ml RB的在一个单独的管中。 </li></ol></li><li>稀释浓缩的配位体的库存到的RB对的〜20微克/毫升的最终浓度。作为一个经验法则，对于稳定的配体的固定化，使用低浓度的配体具有更长的注射在低流量，相对于高浓度，高流量和较短的注射。 </li><li>稀释分析物进入RB至所需浓度。典型地，测试浓度范围为未知亲和力的系统从10微米至10nM的10倍稀释。总是开始与较低浓度的第一。 </li></ol> 2.传感器芯片<ol><li>芯片选择:使用次氮基三乙酸（NTA）适于捕获蛋白质与寡组氨酸标签偶联芯片。 </li><li>芯片的使用: <ol><li>当使用新的芯片，把芯片从鞘，用双蒸水（DDW）轻轻地冲洗，擦干仔细剩余液体用细腻的雨刷器，并确保不要触摸金层的表面。将芯片回其护套以正确的方向（插入是顺畅当芯片被正确放置）。 </li><li>当重复使用芯片，取出从缓冲区存储芯片，具有DDW干彻底冲洗轻轻如上所述，并放入原来的护套。 注:非特异性的垃圾材料的芯片上的累积会影响它的"活力"。这可以通过事先对配体的固定化和后剥离比较的RU进行监测。虽然许多测量可使用相同的芯片NiNTA完成，其长寿的问题不是一个"明确的"，变化很大不同芯片之间。 </li></ol></li><li>芯片对接:打开传感器芯片的门，并把它套芯片。当使用新的芯片，输入在芯片的序列号，以保持使用记录。关好门，然后按&#39;码头芯片"。 </li></ol> 3.温度设置<ol><li>将芯片的细胞的温度至25℃（或优选的实验温度）。 </li><li>可选:设置样品仓至7℃，保持蛋白质整个实验过程中保持稳定。 </li></ol> 4.解决方案加载和脱模准备用于装载和剥离了以下解决方案:为0.5mM的NiCl 2中的RB，350毫摩尔EDTA中的RB（添加洗涤剂至EDTA溶液至终浓度为以RB），0.25％SDS中的H 2 O，和100mM的HCl H中2 O.当使用微型离心机桶ES，而不是SPR指定管，不要忘了切断管帽。 5.总理的SPR仪器与RB 6.启动一个新的运行<ol><li>设置将在实验过程中所使用的流速。以获得这里描述的结果设定流量至50μl/分钟。 注:此参数可在实验过程中进行修改。 </li><li>限定流动通道和参考减法。以获得这里描述设置的路径的Fc = 1和Fc = 2，减法FC = 2-1的结果。 注:该方案将在实时显示两个测量单元的减法。请记住，这个参数不能在实验过程中改变。 </li><li>选择合适的样架（它可以影响样品量消耗）。 </li><li>保存结果文件。 </li></ol> 7.试验周期每个实验由周期，保持EA做了注射清晰记录CH周期，并分离配位体&#39;装载，该缓冲区的空白注射，并且该分析物注射到不同的周期。 <ol><li>周期1:芯片的制备和镍负载<ol><li>确保流动路径设置根据步骤6.1和6.2。 </li><li>注入350毫摩尔EDTA 1分钟洗去剩菜。 </li><li>流量设置为10微升/分钟。 </li><li>注入0.5毫的NiCl 2 2分钟。 注:现在，芯片的树脂是由镍离子涂覆。 </li></ol></li><li>周期2:固定的配体<ol><li>组流动到15微升/分钟，流动路径的Fc = 2。 </li><li>注入配体，直到达到RU值是10-20倍，其分子量。例如，装载的50kDa的配体，以500-1000个RU。不断实现RU值的记录。 </li><li>组流动到15微升/分钟，流动路径的Fc = 1。 </li><li>注入配体B（对照）达到相同的RU值作为配位体A的监视减法sensogram（FC = 2-1）上线，以帮助控制注射长度。 </li><li>集流至50μl/分钟，流动路径的Fc = 1和Fc = 2，洗系统5-20分钟，直到基线（FC = 2-1）稳定。 </li></ol></li><li>周期3:空白注射。该注射将作为空白的减法，因此，包括将与所述分析物被注入，除分析物本身的所有组件。 注意:注射长度可根据需要达到平衡（信号高原）的时间而变化。 <ol><li>组流动到15微升/分钟，流动路径的Fc = 1和Fc = 2，FC = 2-1减法。 </li><li>将"等待"命令（以下简称为"等待"），30秒（有一个稳定的基线）。 </li><li>注入15秒RB。 注:注射的持续时间将不同的系统之间变化，这取决于他们的预平衡动力学常数（主要是K A）。 </li><li>等待120-600秒，记录的解离相。 注意:该步骤的长度根据不同<e米&gt; K D:解离的时间越长这个步骤需要的慢。对于很慢解离性复合物（ 杀敌 &lt;10 -4秒-1），等待10-15分钟，然后进行表面再生（见后）。 </li></ol></li><li>周期4:分析物注射液<ol><li>复制/粘贴到基准喷射（周期3），以便这两个注射以后可以减去使用的确切条件。在从一个持有对照溶液，以一个保持分析物溶液机架改变槽的位置。选择一个浓度稍预期杀敌以上。如果没有先验知识是可用的，选择1μM作为一个很好的起点。 </li></ol></li><li>周期5:缓冲区注入<ol><li>重复循环3。 </li></ol></li><li>周期6:分析物注射液<ol><li>重复循环4。 </li></ol></li><li>周期7:芯片剥离<ol><li>流量设置为50微升/分钟。&lt;/ li&gt; <li>注入350毫摩尔EDTA，1分钟。重复此步骤两次。不要使用3分钟的单次注射，因为这可能会损坏芯片。 </li><li>注入0.25％SDS中1分钟。重复此步骤两次。不要使用3分钟的单次注射，因为这可能会损坏芯片。 </li><li>如果基线水平未达到，注入100毫盐酸，持续1分钟作为附加汽提步骤。 </li><li>插入&#39;端运行"命令，切换到待机模式。 </li></ol></li><li>存储芯片<ol><li>移除传感器芯片，并把它从仪器中。 </li><li>就拿传感器从它的外皮，避免接触到表面，冲洗DDW，并将其放置在50毫升的试管含有RB。保存在4℃。 </li></ol></li></ol> 8.数据分析利用指定的评估软件<ol><li>执行以下步骤来处理动力学参数的推导之前通过SPR获得的原始数据。 <ol><li>使用评估软件，开放结果文件，选择周期3-6 FC = 2-1（参考减去数据）。 </li><li>轴位移: <ol><li>显示周期3-6。 </li><li>零基线（在每个循环中的初始30秒的等待时间），以所有四个曲线的Y轴值。 </li></ol></li><li>对准四条曲线的X轴。选择突出特征（ 例如 ，注射开始或结束的尖峰）到完美地对齐沿X轴的四条曲线。置的分析物（或对照缓冲液）开始到零的时间。 </li></ol></li><li>参考减法<ol><li>减去通过从周期6的曲线中减去周期3的曲线由周期4和周期5的曲线的曲线使用，可以在Y轴中找到曲线减去操作移动菜单背景的响应。 注:它执行此步骤，因为它撤销本任何非特异性组分（无关的分析物），可能影响了表面折射率是重要的。 <ol><li>保存在T减去的曲线他根据不同的名称项目表。 </li></ol></li><li>参阅这些曲线为"双减去曲线":第一减法是在两个注射进行的2-1减法，和第二个是从另一个曲线的减法。 注意:这种双重引用是SPR实验的金标准。 </li><li>通过像以前一样进行解释的光轴偏移的叠加减去曲线。 </li></ol></li><li>动力学常数和模型拟合的决心<ol><li>在进行运动试验<ol><li>注入一系列6-7的分析物浓度，以获得可靠的对动力学常数测定的数据。上述由选择的分析物浓度的10倍至10倍低于估计杀敌 ，在2-3倍稀释。包括"零"的浓度，以后用于双参考减法（见上文）。一式三份，并在随机的顺序进行注射。 </li><li>对齐和减去所有曲线相对于所述Y轴和X轴尝试模型拟合之前。 </li></ol></li><li>模型拟合<ol><li>选择合适的分析程序。 注意:大多数提供装修方案提供可应用于拟合和决心动力学常数的几种模式。 </li><li>应用的最简单的模型（朗缪尔）假设一个可逆1:分析物和配体之间的接头1的相互作用。除非有说服力的数据是从其他的实验方法进行更复杂的相互作用的存在，总是用简单的Langmuir模型。 </li></ol></li></ol></li></ol>

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All authors declare that they have no competing financial interests.

SPR是一种研究分子相互作用高度敏感的方法，并且经常是，它提供了短暂的（但重要）的相互作用的实时监测的唯一方法。一个例子是瞬时相互作用本文中所呈现，不能由任何其他方法（下拉测定法，脂质体沉淀测定法6）来检测。此外，虽然其他方法被限制为平衡测量（不论数量或质量），SPR是衡量也预平衡动力学的唯一技术之一。 其高度的敏感性也是它的警告。?...

蛋白质 - 蛋白质相互作用（PPI）;的形成和蛋白复合物的解离，在许多生物过程（ 如复制，转录，翻译，信号传导，细胞-细胞通讯）的重要事件。通常执行使用下拉或免疫沉淀实验PPI的半定量研究。但是，这些（以及类似）的技术在相关度的可以测量的范围内（低微摩尔和更高的亲和力）由于在洗涤步骤所固有的这些技术的限制。这种"终点"技术不能识别往往是巨大的生物后果短暂的或低亲和力的互动。此外，这种方法的时间分辨率是非常有限的，通常要比反应的速率较慢的订单。 SPR克服了这些缺点，由于其高度的灵敏度和优异的时间分辨率1,2。 SPR是一种无标记方法，因此分子间相互作用可以测量（只要该质量变化，可以检测出）。除了​​PPI，它已被广泛地用于表征蛋白质-配体，蛋白质-药物（或小分子），蛋白质-DNA和蛋白质-RNA相互作用3-5。结果记录并绘制在实际的时间，这使得实验条件和灵活的实验设计的快速修改。 后面的SPR基于票据的物理原理是测量在传感器表面上的折射率的改变时的质量变化2的光学系统的利用率。之一的相互作用伙伴（以下称配体）被固定到聚合物基质芯片和第二分子（以下称分析物）被流过的表面上。分析物结合至该配体改变在芯片表面的质量。这种质量变化是直接按比例变化有关的表面的折射率。该结果绘于实时和呈现为响应单位（RU），为时间的函数。这样的情节被称为sensogram（ 例如 ， 图1）。由于SPR如下的相互作用的完整的时间过程中，预平衡动力学速率常数推导，除了平衡亲和力。如下面详细描述的，一个给定的系统的预均衡行为持有多的信息，并且提供了一个非常不同的角度比单独的平衡。一旦系统被校准，实验是非常快的，并且只需要少量的蛋白材料（微克至纳克量）。收集一个给定的系统的完整动力学信息可以在几天来实现。 SPR的高灵敏度的测量能提供使用的任何其它技术6那是不可能的。然而，这种高灵敏度是一个&#39;双刃剑&#39;，因为它可以为误报数据的主要来源。影响的反射率的任何因素被记录并绘制在sensogram。因此，APpropriate控制必须用来消除误报，并补充办法支持高度建议。 图1示出使用NTA被覆传感器芯片的SPR实验的进展。在面板A中的sensogram示出了喷射的镍离子在NTA基质（未消减数据），并减去从所述阴性对照细胞产生的信号后面板的BD显示数据。红色和蓝色的箭头表示注射的开始和结束，分别。配位体上的芯片固定逐渐改变质量，直到喷射终止。终止配体的注射后观察到的稳定的高原反映配体与所述表面（ 图1B，周期2）的稳定的缔合。一旦一个稳定的基线实现的缓冲器注射在配位体和所述参考单元（ 图1B，周期3）。这注射作为&#39;空白对照"，并且将在分析过程中减去。在注射后，小的变化被记录，反映了质量通过芯片的流动。然后在一个单独的循环（ 图1B，周期4）中，分析物被注入;在RU的逐步增加代表分析物的关联配体。所述结合位点变得逐渐占据直至达到平衡。只要注射结束后，在个RU的下降反映了复杂的解离。从这样的传感图的预均衡和平衡速率常数可以衍生（见后面）。 图1示出了配体和分析物之间的瞬时相互作用。当该相互作用是更稳定的，在RU水平的下降是比较慢，这反映了较低的kð。 这里，我们描述一个SPR实验旨在表征膜转运（洗涤剂溶解）和它的功能性配偶体之间的相互作用，其同源底物结合蛋白6,7。所选择的MOD埃尔系统是一个ATP结合盒（ABC）转运，Archeaglobus fulgidus的ModBC-A。这个系统被选定为它的高度可重复性的结果，高信噪比，和古典&#39;开/关&#39;速率。此外，同源物ABC转运可作为重要的阴性对照。转运，ModBC（配位体A）中，从使用的洗涤剂，纯化和固定到芯片上的膜萃取。其可溶性互动的合作伙伴，MODA，是分析物。作为阴性对照配体，不同的ABC转运RbsBC被使用（"配体B"）。

<table><tbody><tr><td>Biacore T200</td><td>GE Healthcare</td><td>28-9750-01</td><td></td></tr><tr><td>Sensor Chip NTA&amp;nbsp;</td><td>GE Healthcare</td><td>14100532</td><td></td></tr><tr><td>BIAevaluation</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Biacore T200 Software&amp;nbsp;</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Nickel chloride</td><td>Sigma</td><td>N6136</td><td></td></tr><tr><td>Sodium tungstate dihydrate</td><td>Sigma</td><td>T2629</td><td></td></tr><tr><td>Sodium Chloride&amp;nbsp;</td><td>Bio-Lab LTD.</td><td>19030591</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>Ethyldiamine-tetraacetic acid disodium salt dihydrate (EDTA)</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>n-Dodecyl-&amp;beta;-D-Maltopyranoside (DDM)</td><td>Affymetrix</td><td>D310</td><td></td></tr><tr><td>Sodium dodecyl sulfate solution&amp;nbsp;</td><td>Sigma</td><td>05030</td><td></td></tr><tr><td>Kimwipes KIMTECH</td><td>Kimberly-Clark</td><td>34120</td><td></td></tr><tr><td>Hydrochloric acid</td><td>GADOT</td><td>7647-01-0</td><td></td></tr><tr><td>Nylon (NY) Membrane Filter</td><td>Sartorius</td><td>25007--47------N</td><td></td></tr><tr><td>ModBC ABC transporter</td><td>Lewinson lab</td><td></td><td></td></tr><tr><td>RbsBC ABC transporter</td><td>Lewinson lab</td><td></td><td></td></tr><tr><td>ModA Substrate Binding Protein</td><td>Lewinson lab</td><td></td><td></td></tr></tbody></table>

real time measurements of membrane protein:receptor interactions using surface plasmon resonance (spr)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

在本文所描述的系统中，一个NiNTA芯片被用来固定所述His-标记的膜转运6,7。作为同型二聚体，各转运被双重标记，提高其结合到NiNTA芯片。继镍负载，配体A（感兴趣的转运）被固定在FC = 2，多达〜3500 RU（协议周期2， 图2A黑色标签）。然后，用同样的流量和喷射持续时间，配体B（对照配体）被注射到的Fc = 1，最初仅达到多达〜3000 RU。确保相似的蛋白质的量被固定在这两个流?...

Watch this Scientific Journal Video about Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR at JoVE.com

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

表面等离子共振（SPR）是一种无标记检测方法，实时生物分子相互作用。在这里，协议的膜蛋白:受体相互作用实验说明，在讨论该技术的优点和缺点。

膜蛋白的实时测量:受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

real-time-measurements-membrane-proteinreceptor-interactions-using

Research

JoVE Journal

Biology

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

22.6K 次观看.  The Technion-Israel Institute of Technology. 表面等离子共振（SPR）是一种无标记检测方法，实时生物分子相互作用。在这里，协议的膜蛋白:受体相互作用实验说明，在讨论该技术的优点和缺点。 

视频: Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

科研

生物化学

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

生物工程

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

生物膜荧光探针力:受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

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. ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

生物学

Real-Time Detection of Dynamic Affinity between Biomolecules Using Surface Plasmon Resonance (SPR) Technology

Surface plasmon resonance (SPR) technology is a sensitive precise method for detecting viruses, pathogenic molecular proteins, and receptors, determining blood types, and detecting food adulteration, among other biomolecular detections. This technology allows for the rapid identification of potential binding between biomolecules, facilitating fast and user-friendly, non-invasive screening of various indicators without the need for labeling. Additionally, SPR technology facilitates real-time detection for high-throughput drug screening. In this program, the application field and basic principles of SPR technology are briefly introduced. The operation process is outlined in detail, starting with instrument calibration and basic system operation, followed by ligand capture and multi-cycle analysis of the analyte. The real-time curve and experimental results of binding quercetin and calycosin to KCNJ2 protein were elaborated upon. Overall, SPR technology provides a highly specific, simple, sensitive, and rapid method for drug screening, real-time detection of related pharmacokinetics, virus detection, and environmental and food safety identification.

Real-Time Detection of Dynamic Affinity between Biomolecules Using Surface Plasmon Resonance SPR Technology

The present study aims to elucidate the principle and methodology of surface plasmon resonance (SPR) technology, which finds versatile applications across multiple domains. This article describes SPR technology, its operational simplicity, and its remarkable efficacy, with the goal of fostering broader awareness and adoption of this technology among readers.

Surface plasmon resonance (SPR) technology is a sensitive precise method for detecting viruses, pathogenic molecular proteins, and receptors, determining ...

医学

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

多路复用表面等离子体共振成像芯片上的多模态分析平台，用于分析细胞外囊泡亚群

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) is the underlying optical phenomenon behind label-free biosensors to evaluate the molecular affinity, kinetics, specificity, and concentration of biomolecules. In SPR, biomolecular interactions occur on a biosensor made of a thin layer of metal on a prism. Real-time interactions of biomolecules can be monitored by measuring the changes of light reflected off the underside of the metal.
This video describes the basic concepts of SPR and how it is used to analyze and visualize biomolecular interactions. This is followed by a sample preparation and experimental protocol for investigating binding rates using SPR. In the applications section, SPR imaging, localized SPR, and quantum dot enhanced SPR are explored.
<div class="chapter_text" id="script_0">
Surface plasmon resonance, or SPR, is the underlying phenomenon behind certain label-free biosensors for evaluating binding and adsorption interactions of biomolecules. Binding assays that require labeling, such as ELISA, can be a time-consuming process, and may alter the functionality of the analyte. In SPR, biomolecular interactions occur on a special sensor made of a thin layer of metal on one face of a prism. By monitoring the changes in light reflected off of the underside of the metal, SPR instruments visualize these interactions in real-time without the use of labels. This video will introduce the principles of SPR, a general procedure for SPR imaging, and some applications of in biochemistry.
</div>
<div class="chapter_text" id="script_1">
An SPR sensor is usually made of a thin layer of a noble metal atop the face of a prism. To take readings from the sensor, light is reflected off of the prism-metal interface into a photodetector. The reflected light will have a high intensity except at a certain angle, related to the electronic properties of the metal surface, known as the &#34;surface plasmon resonance angle&#34;.
As molecules bind to the surface, the electronic properties of the metal change, which in turn adjusts the angle. As new proteins attach, forming complexes, the angle will shift further. By measuring relative changes in the SPR angle, interactions like these can be monitored in real-time.
Another technique called localized, or &#34;L&#34;SPR, uses metal nanoparticles as the sensor surface. The properties that affect the SPR angle are highly localized to each nanoparticle, which improves sensitivity and signal resolution.
When investigating binding interactions with standard SPR, the sensor is generally mounted in a platform that becomes the floor of a flow cell in the instrument. The biomolecules of interest are carried through the flow cell by buffer solution. The sensor surface is often first coated with a substrate that has a high affinity for the metal. This ensures that a significant amount of ligand, which in turn binds to the analyte of interest, will be immobilized onto the sensor and reduces the likelihood that the ligand will dissociate during the procedure.
Once the ligand is immobilized on the sensor, the analyte is flowed over the sensor in buffer. By monitoring the change in the SPR angle over time as the analyte binds to the ligand, the binding rate and other kinetic information can be calculated.
The reflectance data can also be used for SPR imaging, or SPRi, by directing the reflected light to a CCD detector. This produces a high-contrast, high-resolution image of the entire sensor surface. Using SPR and the related techniques, questions can be answered about molecular affinity, kinetics, specificity, and concentration.
Now that you understand what is being measured in an SPR experiment, let&#39;s look at a procedure for investigating binding rates.
</div>
<div class="chapter_text" id="script_2">
Before beginning the procedure, the running and sample buffers must be prepared. The running buffer is used to deposit the ligand onto the sensor, and the sample buffer is used to deposit the analyte. The sensor chip is carefully cleaned and loaded into a sheath. Then, the device is placed into the instrument, where it becomes the bottom of the flow cell. The instrument software is set up for the experiment and subsequent analysis. If necessary, the sensor surface is primed with a substrate to capture the ligand. The ligand is flowed over the sensor surface in the running buffer, where it is captured by the substrate on the sensor surface.
Then, the analyte in the sample buffer is run through the flow cell, where it selectively binds to the immobilized ligand. The change in reflectance is plotted and compared against controls to determine rate constants and other reaction kinetics data for the investigated reaction.
</div>
<div class="chapter_text" id="script_3">
Now that you understand how an SPR experiment is performed, let&#39;s look at a few other applications of SPR in biochemistry.
Here, SPR imaging was used to evaluate proteins with an array of eleven receptors on a sensor. 3D graphs of reflectivity versus time and receptor concentration were prepared from the reflectivity data for each protein. These &#34;profiles&#34; are characteristic to each protein, and thus could subsequently be used for protein identification.
In this experiment, cell secretions were studied using a custom-made LSPR sensor. The sensor was also compatible with SPRi and fluorescence microscopy. Upon depositing the cell on the sensor, the interaction of cell secretions with the nanoparticle array could be measured with high spatial resolution.
Here, the use of quantum dots, nanoscale semiconductors, as an SPR signal enhancement agent mixed with the analyte was investigated. This enhanced &#34;nano-SPRi&#34; method was compared to assays by standard SPRi and the ELISA method. The nano-SPRi method significantly improved the sensitivity and limit of detection while still being less time-consuming than the ELISA method.
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You&#39;ve just watched JoVE&#39;s video on surface plasmon resonance. This phenomenon is used to monitor and image biomolecular interactions without the use of labels. This video introduced the principles of SPR, a typical protocol for performing an SPR experiment, and a few applications of SPR in biochemistry.
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Surface Plasmon Resonance SPR: Label-Free Detection

表面等离子体共振 (SPR)

Surface plasmon resonance (SPR) is the underlying optical phenomenon behind label-free biosensors to evaluate the molecular affinity, kinetics, ...

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Surface Plasmon Resonance to Study Biomolecular Interactions Using a Sensor Chip

Source: Maier, I. <a href="https://app.jove.com/v/63541">Engineering Antiviral Agents via Surface Plasmon Resonance.</a> J. Vis. Exp. (2022)
This video describes the surface plasmon resonance, or SPR, technique for studying the interaction between two molecules using a sensor chip. The change in resonance angle and the intensity of the reflected light are detected as a function of the association and dissociation of two molecules, and this provides information about the interactions between the two molecules.

表面等离子体共振，使用传感器芯片研究生物分子相互作用

Source: Maier, I. Engineering Antiviral Agents via Surface Plasmon Resonance. J. Vis. Exp. (2022)
This video describes the surface plasmon resonance, or ...