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”）。

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			<td height="42" style="height:42px;width:209px;">Name of Material/ Equipment</td>
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			<td height="21" style="height:21px;">Biacore T200</td>
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			<td height="21" style="height:21px;width:209px;">Sensor Chip NTA&#160;</td>
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			<td height="21" style="height:21px;width:209px;">BIAevaluation</td>
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			<td height="21" style="height:21px;width:209px;">Biacore T200 Software&#160;</td>
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			<td height="21" style="height:21px;width:209px;">Sodium tungstate dihydrate</td>
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			<td style="width:119px;">T2629</td>
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			<td height="21" style="height:21px;width:209px;">Sodium Chloride&#160;</td>
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			<td height="63" style="height:63px;width:209px;">Ethyldiamine-tetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">E5134</td>
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			<td height="41" style="height:41px;width:209px;">n-Dodecyl-&#946;-D-Maltopyranoside (DDM)</td>
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			<td height="42" style="height:42px;width:209px;">Sodium dodecyl sulfate solution&#160;</td>
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			<td height="21" style="height:21px;width:209px;">Kimwipes KIMTECH</td>
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			<td height="21" style="height:21px;width:209px;">ModBC ABC transporter</td>
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			<td height="21" style="height:21px;width:209px;">RbsBC ABC transporter</td>
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			<td height="42" style="height:42px;width:209px;">ModA Substrate Binding Protein</td>
			<td style="width:107px;">Lewinson lab</td>
			<td style="width:119px;"></td>
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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)

22.5K 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

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

科研

生物学

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

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 ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

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 ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...