这项研究是由ISF，NSF，和ERC。我们要感谢技术帮助，兰迪·米尔福德，迈克尔·科伦，道格·谢弗和杰里米·丹尼森的温度阶段。或陈，徐迪，拉杰什Sannareddy，和SUMIT Bhattachary与软件开发提供了援助。我们要感谢我们的合作者教授彼得·戴维斯博士和劳瑞A.格雷厄姆的熔点的 IBP蛋白质和有益的讨论。我们也感谢实验室成员玛雅酒吧DOLEV，扬中市秦，Yeliz切利克博士，博士纳塔利娅Pertaya，Ortal Mizrahy，并为他们的用户反馈Shlomit盖伊博士。

0。初步程序<ol><li> 玻璃毛细管中的溶液注入。，使用毛细管牵拉（成茂，东京，日本）中，准备一个尖锐的吸移管，具有细小开口的玻璃毛细管的微管（品牌GMBH，韦特海姆，德国）。的开口的大小应加以核实通过空气通过毛细管，得到细的干净的水鼓泡。如果毛细管被关闭，然后打开它，打破它的优势。这可以通过对水中含有管壁它轻轻地按压或刮伤。准备的毛细管，使得开口几乎是阻止，但被充分地打开，以允许亚毫米的气泡的形成。 </li><li> 铜光盘清洁。超声波清洗铜光盘在0.1％微-90肥皂（Cole-Parmer公司，弗农山，伊利诺斯州，美国）的10分钟，然后用双蒸水清洗。异丙醇（技术）解决方案和介绍光盘，再次超声处理10分钟。鳍盟友，干燥的光盘使用过滤后的空气。这个清洁阶段中是至关重要的，以避免的IBP污染之间实验。 </li><li> 双层玻璃罩装配，一个玻璃罩装配，准备让无盖玻璃表面的水分冷凝样品的观察。这是通过两个盖玻片上，然后将胶与热胶枪之间放置一个燥石膏（WA哈蒙德燥石膏，捷尼亚，俄亥俄，美国）颗粒（直径2 mm）。这种配置防止冷凝，当样品被冷却至低温并除去吹干燥空气中的观察窗上的需要，可以阻止视图。 </li></ol> 1。冷却阶段， <ol><li>在冷却阶段的水的流入口和出口连接到4毫米内径的Tygon管（圣戈班，巴黎，法国），和水流入口管连接到一台水泵。 </li><li>一个4毫米内径的Tygon管连接到入口的冷却阶段tØ提供干燥的空气。的空气干燥使用一个在线燥石膏列。 </li><li>操作空气和水的泵。请注意，冷却的元素不应该被未经散热器运行。 </li><li>打开温度控制器，摄像头，和LabVIEW常规。 </li></ol> 2。样品制备<ol><li>放置浸油B（0.0004实验室锡达格罗夫，新泽西州，美国）上的背面侧的直径7mm的铜盘具有500μm的钻孔通过盘3-4微升液滴。 </li><li>铜盘定位面朝下浸油的冷却阶段。 </li><li>连接的毛细管（钝缘）0.7毫米内径的Tygon管的另一端连接到一个2毫升玻璃的注射器（Poulten-格拉夫，韦特海姆，德国）。 </li><li>使用毛细管之前，检查所述毛细管的小开口，以确保该开口是一个适当的大小（见初步的程序）。 </li><li>慢慢地将冰川SS毛细管进入准备IBP蛋白质样品管（2.4μM的熔点 IBP-GFP在20mM的CaCl 2和25mM的Tris-HCl在pH值为8，用于制备细节，见参考文献10）和拉玻璃注射器，直到玻璃毛细管含有0.1微升的蛋白质溶液。 </li><li>通过LabVIEW软件，开始录像记录。 </li><li>的玻璃毛细管（含蛋白质溶液）成铜光盘上的冷却阶段中的孔之一插入的锋利边缘。 </li><li>当通过显微镜观察（奥林巴斯，日本，东京，10x物镜），小心地渗透层与玻璃毛细管尖端的浸油，并按下的玻璃注射器（非常微妙），以提供一个小的量的蛋白质（〜10 nl）的解决方案来创建一个200微米的液滴。 </li><li>在冷却阶段组件（见初步程序）与双层盖玻片覆盖孔。 </li></ol> 3。 TH活性的测定<ol><li>前SS的冷却按钮，设定温度为-40°C。 </li><li>最初，该溶液液滴将被清除。在低温下，通常在-30℃至-35℃下，液滴的颜色会发生变化，这表明该溶液已被冻结。样品后立即冻结，慢慢地提高温度，直到大量的冰开始融化。的温度会逐渐增加是必要的，以避免过冲的，可能会导致在完全熔化的样品的温度。 </li><li>切换到50倍的目标，并通过调节温度开始融化的冰。这种调整是交互式的，0.002℃，用小的温度的步骤，通常执行的最后步骤继续融化，直到单晶仍然存在。晶体的最终大小应该是大约10微米。的最高温度，在该温度下熔化已不再被确定为熔点，并精确地确定在购买的视频分析阶段。 </li> &lt;李&gt;把温度设置为几百分之一摄氏度的熔点以下的晶体，并用10分钟的延迟开始的温度斜坡。调整所需的升温速率。在这段时间内，晶体将被暴露的IBPS。 </li><li>完成后的10分钟的曝光时间，温度会的LabVIEW例程的控制下，自动降低。 </li><li>观察晶体形状随着温度的降低。在某些时候，突然一阵冰晶可以观察到。注意到作为晶体突发温度的温度，在这温度下，发生这种情况。 </li><li>使用视频分析，以确定准确的熔点和脉冲串的温度。首先，通过使用视频分析，找到准确的熔点。回想一下，最高温度，在该温度下熔化已不再被确定为熔点。记下这个熔点在电子表格程序。然后，确定准确的结晶的爆破温度，并记录该值为好。的熔点和冰点，或晶体突发温度之间的差异是的IBP溶液的热滞后活性。 </li></ol> 4。的依赖于时间的TH活动测量<ol><li>按照协议第3.1-3.3准备一个冰晶体。 </li><li>晶体形成后，设定所需的延迟时间的坡道，以及打开的坡道上。 </li><li>温度将降低固定利率（根据运营商的要求）后，自动坡道延迟时间已经过了。 </li><li>记录的晶体突然发生的温度，在该温度下。计算的曝光时间（晶体的形成和晶体的脉冲串之间的时间）。 </li><li>重复实验，不同的延迟时间，和作为曝光时间的函数绘制的TH活性评估TH活性的时间依赖性。 </li></ol>

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没有利益冲突的声明。

这项工作表明操作的计算机控制的纳升渗压计的TH活性具有非凡温度控制，使精确的测量。在任何温度敏感的系统中，必须避免不必要的温度梯度。为了避免在这里提出的装置中的温度梯度，在测试溶液液滴必须被定位在铜圆盘冷却阶段（步骤2.7）中的孔的中心。此外，单晶应该是在液滴的中心，而不是靠近边缘（在大多数情况下，这会发生自发）。描述的时间依赖关系表示的冷却速率可能会影响对TH读数。因此，我们建议包括一个报告的时间，在此期间，晶体被暴露到该溶液中，在冷却之前，以及冷却速度。通常，我们等了10分钟前，斜坡下降的温度为0.01°C步骤，每4秒。 LabVIEW的控制合作适于使用倒置显微镜，微流体装置可热操纵oling阶段。此系统有利于涉及冰晶和eGFP的9，10，16具有标记IBPS溶液交 ​​换实验的性能。 LabVIEW的控制系统可适于到克利夫顿阶段通过连接3040通过一个指定的自适应电路的温度控制器。这样一个系统的操作在戴维斯的实验室17。 LabVIEW软件和指定的克利夫顿阶段的适应电路设计可应要求提供。 总之，我们描述了纳升渗压计，有利于敏感的控制和操作的温度和温度的升高和降低的速度（0.002°C灵敏度），配合视频接口，通过LabVIEW的程序进行实时分析。该系统可以进行重复的速率控制实验，是的importan调查IBP与冰相互作用的动力学吨。这样的实验可以解决一些长期争论的问题IBPS行动的机制。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号/型号 </td><td> 评论 </td></tr><tr><td>浸油B型</td><td> 0.0004实验室</td><td> 16484 </td><td></td></tr><tr><td>燥石膏</td><td>华盛顿州哈蒙德燥石膏</td><td> 0430632270克</td><td></td></tr><tr><td>微90的清洗液</td><td> Cole-Parmer公司</td><td> <a href="http://www.coleparmer.com/Product/Cole_Parmer_small_sup_reg_sup_small_Micro_90_small_sup_reg_sup_small_cleaning_solution_case_of_four_4_Liter_bottles/EW-18100-11" target="_blank">EW-18100-11</a> </td><td></td></tr><tr><td>毛细管拉马</td><td>成重</td><td> PB-7 </td><td></td></tr><tr><td>玻璃毛细管</td><td>品牌GNBH </td><td> 7493 21 </td><td> 75毫米长，直径1.15 </td></tr><tr><td>温度调节器</td><td>新港，尔湾，加利福尼亚州，美国</td><td>型号3040 </td><td>型号3040 </td></tr><tr><td>光学显微镜</td><td>奥林巴斯</td><td>型号BH2 </td><td></td></tr><tr><td> 10倍的目标</td><td>奥林巴斯</td><td></td><td>的计划，0.3％，160/0.17 </td></tr><tr><td> 50倍的目标</td><td>尼康</td><td></td><td> CF计划，50X/0.55 EPI ELWD </td></tr><tr><td> CCD相机</td><td>丕</td><td> CVC-140 </td><td></td></tr><tr><td>聚乙烯管</td><td>巴黎，法国圣戈班， </td><td></td><td>的Tygon配方S-50-HL油管</td></tr><tr><td>玻璃注射器（2毫升） </td><td> Poulten格拉夫，韦特海姆，德国</td><td> 7 10227 </td><td></td></tr><tr><td> GPIB-PCI卡</td><td>国家文书，美国得克萨斯州奥斯汀​​， </td><td> 778032-01 </td><td></td></tr><tr><td>视频帧GRABBER IMAQ PCI-1407 </td><td>国家文书，美国得克萨斯州奥斯汀​​， </td><td> 322156B-01 </td><td></td></tr><tr><td> LabVIEW系统设计软件</td><td>国家文书，美国得克萨斯州奥斯汀​​， </td><td>第8版</td><td></td></tr><tr><td>的DIVX作者软件</td><td> DIVX，圣迭戈，CA，USA LLC </td><td></td><td></td></tr></tbody></table>

labview-operated novel nanoliter osmometer for ice binding protein investigations

冰结合蛋白（IBPS），包括抗冻蛋白，冰结构蛋白，热滞蛋白，冰结晶抑制蛋白，被发现在冷适应的有机体，并保护它们与冰晶冷冻损伤。 IBPS被发现在各种有机体，包括鱼1， ​​植物2，3，节肢动物4，5 6，真菌和细菌7。 IBPS吸附到冰晶体的表面，并防止水分子从加入冰格在IBP吸附位置。制冰上生长的晶体表面之间的吸附IBPS开发高曲率，降低温度，在该温度下的冰晶体的成长，作为为Gibbs-Thomson效应提到的现象。此抑郁创建的熔点和冰的生长，在其内被逮捕8-10，参见图1的非平衡凝固点之间的间隙（热滞后，TH）。上IBP研究中使用的主要工具是e的纳升渗压计，这有利于测量TH活动的IBP解决方案。纳升渗压计，如克利夫顿仪器“（克利夫顿技术物理系，哈特福德，纽约州）和奥塔哥仪器”（奥塔哥渗压计，达尼丁，新西兰），设计用于测量溶液的渗透压测量的熔点抑郁症的液滴纳升卷。这些设备被用来测量生物样品，如眼泪11的渗透压，并在IBP研究发现是有用的。这些纳升渗压计的手动控制限制了实验的可行性。温度变化率的变化，无法可靠地进行控制，克利夫顿仪器的温度范围内被限制到4000毫渗透摩尔（约-7.5℃），并作为时间的函数的温度记录这些仪器不是可用的选项。 我们设计了一个特制的计算机控制的南欧力特渗压计系统使用LabVIEW平台（美国国家仪器公司）。冷阶段，前面描述的9，10，包含一个金属块，水通过该循环，从而起到作为散热器，请参阅图2。附加到该块是可以使用商业的温度控制器，该控制器可以控制通过LabVIEW模块驱动的热电冷却器，请参阅图3。下面提供进一步的细节。该系统的主要优点是它的感温控制， 见图4。自动温度控制，允许一个固定的温度斜坡的协调与视频显微镜输出包含更多的实验细节。 研究TH活动与时间的关系，我们测试了58 kDa的多动IBP从南极的细菌Marinomonas primoryensis（MP IBP）12。这种蛋白质有利于增强型绿色荧光标记蛋白（EGFP）在一个结构开发的彼得·戴维斯组（皇后大学）10。我们发现，受影响的温度变化曲线TH活动。在这些实验中的温度分布的极好的控制显着改善了对TH测量。纳升渗压计，还使我们能够测试IBPS 5星，13的重结晶抑制。在一般情况下，重结晶是一种现象，其中大的晶体生长在牺牲小晶体大。 IBPS有效地抑制再结晶，即使在低浓度14，15。我们使用LabVIEW控制的渗压计，定量跟踪冰的再结晶和执行恒定的冰部分使用的同步实时视频分析的图像和温度反馈，从样品室13。实时计算提供了更多的控制选项，在实验过程中。倒置显微镜发展到一个阶段ccommodate温度控制的微流体装置，这将在其他地方描述16。 冷战阶段系统冷阶段的组件（ 图2）包括一组热电冷却器，冷却铜板。热量被从阶段通过流动冷水通过一个封闭的室下的热电冷却器除去。 4毫米直径的孔，在中间的铜板作为观看窗口。直径1毫米的平面内孔，钻孔，以适应热敏电阻。甲量身定制的铜与几个孔（直径500微米）的光盘（7毫米的直径）的铜板上，并置于与观看窗口对齐。泵入空气的流速为35毫升/秒，并干燥，用燥石膏（华盛顿州哈蒙德）。使用的干燥空气，以确保干燥的环境中，在冷却阶段。该阶段是通过一个9针连接插座连接温度控制器（型号3040或3150，Newport公司尔湾，加利福尼亚州，美国）。温度控制器通过电缆连接到计算机的GPIB-PCI卡（美国国家仪器，奥斯汀，德克萨斯州，美国）。

测量TH时间依赖性 LabVIEW的操作纳升渗压计，有利于性能测量准确TH的活动。恒定的温度降低率允许的TH的时间依赖性的测量。精确的温度控制使能纳升渗压计，这些实验是至关重要的。冰晶体在溶液中的IBPS的曝光时间被定义为形成的晶体（熔化过程的结束）的时间周期从直到周围的冰晶体（晶体突发）的突然增长。我们发现，曝光时间的冰晶体的IBPS关键影响TH活性。短时间的的IBP曝光（几秒钟）生产的低TH活动在 MP IBP-GFP溶液（2.4μM）（ 图5）。 TH活性增加与IBP曝光时间，直到它到达一个高原在4分钟IBP曝光。在较高的初馏点浓度，该板非盟在较短的时间内达到。 <img alt="图1" src="/files/ftp_upload/4189/4189fig1.jpg" /> 图1的示意图示出IBPS吸附到冰。通过许可10。 <img alt="图2" fo:src="/files/ftp_upload/4189/4189fig2highres.jpg" src="/files/ftp_upload/4189/4189fig2.jpg" /> 图2的冷却阶段。 A）连接到管显微镜。 B）没有上引线。 C）示意图。 <a href="http://www.jove.com/files/ftp_upload/4189/4189fig2large.jpg" target="_blank"> </a><img alt="图3" src="/files/ftp_upload/4189/4189fig3.jpg" /> 图3。的LabVIEW界面的屏幕截图。 <a href="http://www.jove.com/files/ftp_upload/4189/4189fig3large.jpg" target="_blank">氯</a>ICK这里查看大图。 <img alt="图4" fo:src="/files/ftp_upload/4189/4189fig4highres.jpg" src="/files/ftp_upload/4189/4189fig4.jpg" /> 图4的温度稳定性的图。该温度控制器被设置为较低的温度0.01°C每15秒。 <img alt="图5" fo:src="/files/ftp_upload/4189/4189fig5highres.jpg" src="/files/ftp_upload/4189/4189fig5.jpg" /> 图5， 熔点 IBP TH活性作为冰晶曝光时间的函数的IBPS。每个时间点是3-6次实验的平均值。

Watch this Scientific Journal Video about LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations at JoVE.com

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

冰结合蛋白（IBPS），也被称为抗冻蛋白，抑制冰的生长及组织的冷冻保存中使用的是一个有希望的添加剂。调查IBPS使用的主要工具是纳升渗压计。我们开发了一个设计的冷却阶段安装在光学显微镜和控制使用一个自定义内置的LabVIEW常规。这里所描述的纳升渗压计在一个超灵敏的操纵样品的温度。

LabVIEW操作的新型纳升渗透压仪冰结合蛋白的调查

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

labview-operated-novel-nanoliter-osmometer-for-ice-binding-protein

Research

JoVE Journal

Chemistry

20.3K 次观看.  The Hebrew University of Jerusalem. 冰结合蛋白（IBPS），也被称为抗冻蛋白，抑制冰的生长及组织的冷冻保存中使用的是一个有希望的添加剂。调查IBPS使用的主要工具是纳升渗压计。我们开发了一个设计的冷却阶段安装在光学显微镜和控制使用一个自定义内置的LabVIEW常规。这里所描述的纳升渗压计在一个超灵敏的操纵样品的温度。

视频: LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.

表面钝化单分子蛋白质研究

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important ...

科研

化学

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

BMP-2的共价结合利用表面上的自组装单层膜法

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

通过荧光基冰平面亲和力确定防冻蛋白的冰结合平面

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

生物层干涉测量的蛋白质 - 蛋白质相互作用和变构配体反应动力学的影响

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

铱（III）荧光探针检测疟疾蛋白质生物标志物组氨酸丰富的蛋白质-Ⅱ

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

水在油乳剂：与疏水性颜料装配水溶性叶绿素结合蛋白的新系统

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

的ELISA基结合和竞争法快速测定配体 - 受体相互作用

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

CN-GELFrEE  - 透明天然凝胶洗脱液体馏分偷天陷阱电泳

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

在EOB-DTPA和镓（三）复杂的调查其<sup&gt; 68</sup&gt;嘎放射性标记模拟

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

观测 Photoresponsive 液晶结构动力学的新方法

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...