我们感谢FortéBio用于提供在图1中所用的图形。这项工作是由美国国立卫生研究院授予GM083088以支持战区导弹防御系统

<html> 1。对仪器进行编程的BLI分析打开仪器至少一小时提前让灯泡预热，这是必要的，以减少噪音和实验过程中漂移的光信号。通过仪器标签设定所需温度，以prewarm样品板支架。然后设置实验设计中的数据采集软件。在实验向导选项卡中选择“新建动力学实验”。这给那些必须在定义的所有步骤的选项卡式菜单。 1.1。板定义定义列要在96孔样品板使用。指定列包含缓冲，固定蛋白质或约束力的合作伙伴。对于每个协会以及输入绑定的合作伙伴所使用的浓度。注意：在图2所示的板定义为具有不同的试验方案。 1.2。法定义ontent“&gt;定义所需的检测的所有步骤。这些包括：（i）基线（若干），（二）装载，（三）协会和约束力的合作伙伴（四）解离，然后，如下所述，链接各个实验步骤，在样品板的孔中通过选择在相应的列中的检测步骤，然后双击之列。此方案的传感器就可以从孔中的一列在试验过程中移动到下一个。 <ol><li> 底线 首先简要基线的步骤（≥60秒），并在测定缓冲液中的传感器（ 图2中 ，第1列），以建立初始BLI信号在96孔板中。 </li><li> 中 （固定在传感器上的生物素化的蛋白质） 向孔中，将含有生物素化的蛋白（ 图2，第2列），分配的传感器。使用阈值函数来实现的结合预定的水平（见简介）。设置阈值选项，这样所有的传感器将举d，来井的下一列时，传感器中的任何一个达到阈值。 </li><li> 底线 包含在缓冲区中的另一基线的步骤（ 图2，第3列;通常&gt; 5分钟），以从传感器洗去未固定的生物素化的蛋白质，并建立新的，稳定的基线信号。对于基本的结合/解离测定仅（ 如图3），包括前协步骤与在缓冲液中的新井中的传感器一个额外的基线的步骤（60秒）（ 图2，第4栏）。 </li><li> 协会 （绑定合作伙伴，固定化蛋白质） 对于一个基本的结合/解离吸附测定（如在图3），选择的范围内结合配偶体的浓度以用于不同的传感器/孔（ 图2，第5列）。调整步骤的时间，使得至少一个饱和的结合配偶体的浓度应接近平衡结合信号。对于实验测试的小配体对蛋白质-蛋白质复合物（ 如图5）的解离变构效应，选择一个高浓度的具有约束力的合作伙伴（〜10倍以上，估计杀敌D）在所有关联井使用。 </li><li> 解离 （绑定合作伙伴） 对于一个基本的结合/解离测定仅（ 如图3），指定该传感器返回到第4列（ 图2），作为用于协会之前的额外基线的相同缓冲液的孔中。对于一个测定测试的小配体的变构效应，而不是指定的传感器移动到第6列（ 图2），其中，每个孔可以包含缓冲液加不同的变构配体。 </li></ol> 1.3。传感器分配表明在传感器托盘将包含预湿传感器，用于测定的位置。识别任何空位置，因为该实验将辉l如果没有传感器被拾起。 1.4。回顾实验可视化所有计划的步骤来检查错误并返回到纠正。 1.5。运行试验输入必要的细节，包括数据文件的位置。输入的实验所需的温度。如果传感器仍需要预湿，选择延迟在实验开始的选项。最后，当所有的样品制备完成后（步骤2），这两个样品板和传感器托盘在仪器被加载，点击Go按钮来运行检测。 2。样品制备<ol><li>准备一个合适的分析缓冲液。缓冲器用于在图3和图5所示的实验：20mM的MOPS（3 - （N-吗啉）丙磺酸），Tris（三（羟甲基）氨基甲烷）（加入以调节pH至8.0），50mM的氯化钾。包含BSA（牛血清白蛋白，脂肪酸游离，0.5mg / ml的终浓度）在缓冲液中的所有测定法的步骤和对生物素化的蛋白或结合配偶体，以尽量减少蛋白的非特异性结合到传感器的所有稀释液。 </li><li>在测定缓冲液中稀释的生物素化的蛋白质（ε），以适当的浓度（参见引言）。 </li><li>准备结合合作伙伴（F 1（-ε））的分析缓冲液（见讨论的浓度使用范围）的稀释液。 </li><li>预湿的链亲和素包被的传感器，至少10分钟，以去除其保护蔗糖涂层。从传感器托盘中取出传感器内置型架以及在托盘的底部插入一个96孔板中，在托盘上滑动的板的一个角向定向槽口。对于要使用的传感器的列中，在96孔板中的该列中添加200微升测定缓冲液，每孔。返回传感器的机架在正确的方向托盘（带选项卡的插槽）。 </li><li>如步骤1在编程过程中分配，填写井ØF中的样品板与测定缓冲液中或相应的蛋白稀释液，如在图2中 。避免引入气泡，因为它们会导致噪音的光信号中。包括一个或多​​个基准井省略或者（i）生物素化的蛋白质或（ii）结合配偶体。 </li><li>打开仪器的门，将传感器托盘走上舞台（左），与插入的阶段插槽托盘的标签。插入样品板到所述板支架（右）;确保板落座平面和以正确的方向，作为该板夹持器上表示。关上门，从数据采集程序（步骤1.5）开始检测。 </li></ol> 3。数据处理<ol><li>该试验运行后，打开数据分析软件，并加载包含数据的文件夹。点击“处理”选项卡中看到一个逐步处理菜单（左）和原始动力学数据，每个传感器（AH）分配不同的颜色（参见图3）。 </li><li>在“数据选择”，点击“传感器选择”按钮。在“样品板图”，指定为参考井的井为1或2控制传感器（G，H在图3的分析）。在Processing菜单，勾选“减法”框中，选择“参考井”，从每一个其他传感器的信号中减去参考信号（单个或平均值）。 </li><li>通过使用“对齐Y轴”步骤中的所有痕迹对齐到Y = 0。选择“基线”作为对准步骤（这是之前的关联步骤的最后底线）。对于“时间范围”，进入最后10秒的基线（ 即 50〜60秒，60秒的基线， 如图三 ）。 </li><li>勾选“跨一步修正”框，以尽量减少该协会与解离步骤之间的信号变化。选择对齐基线或解离。 </li><li>在大多数情况下，选择Savitzky-Golay滤波功能，然后单击“进程的数据！”以继续。目视检查最终处理的数据（右下图）。随着检测用于全局数据分析（ 如图4），如果信号迹线显示协会及分解步骤之间有显著转变，改变解离或基线为“跨一步修正”的评选，并进行到以前的数据重新处理数据分析。 </li></ol> 4。数据分析点击“分析”选项卡中数据分析开始。注意：下面的例子中的步骤是对多个结合/解离曲线全局分析（ 如图3）。 <ol><li>对于“步骤来分析”，选择结合和解离。为“Model”，选择1:1。注：其他有限选项可供选择。 </li><li>对于“拟合”，选择环球（完整版）。对于“分组依据”中选择颜色（如在图上）。选择“R 最大未链接到传感器”，让R 最大的独立配件（饱和后面值的约束性最大的信号响应tner与固定化的蛋白）。注：我们这样做对大多数检测，如传感器略有不同蛋白质的固定化量（ 见图3，加载）。 </li><li>上所示的表，确保所有传感器的痕迹进行分析具有相同的颜色，所以他们会作为一个全球性集进行分析。如果需要，选择一个或多个传感器的痕迹，从全局拟合省略：在“包含”一栏，所需的传感器位置单击鼠标右键，选择“排除井”。 </li><li>点击“拟合曲线”！启动非线性回归分析。检查拟合结果，其中包括与传感器的数据痕迹回归曲线（ⅰ）的覆盖（ 如图4），（ⅱ）重复拟合的残差，以及（iii）与所确定的参数值（速率常数，R 最大的表， 杀敌D）和统计（参数标准误差，卡方检验，R 2）。 </li><li>在“数据导出”，单击“导出表到。csv文件”拯救拟合结果。对于GRAP兴或与其他软件的数据/拟合曲线进一步分析，单击“导出拟合结果”来保存每个传感器的数据和拟合曲线在一个文本文件中。 </li></ol>

<ol>
	<li>Nirschl, M., Reuter, F., Voros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+Transducer+Principles+for+Label-Free+Biomolecular+Interaction+Analysis.">Review of Transducer Principles for Label-Free Biomolecular Interaction Analysis.</a> Biosensors. 1, 70-92 (2011).</li><li>Homola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+and+future+of+surface+plasmon+resonance+biosensors.">Present and future of surface plasmon resonance biosensors.</a> Anal. Bioanal. Chem. 377, 528-539 (2003).</li><li>Piehler, J., Brecht, A., Gauglitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+Detection+of+Low+Molecular+Weight+Analytes.">Affinity Detection of Low Molecular Weight Analytes.</a> Anal. Chem. 68, 139-143 (1996).</li><li>Duncan, T. M., Hackney, D. D., Tamanoi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Enzymes.">The Enzymes.</a> Energy Coupling and Molecular Motors Vol. XXIII, 203-275 (2004).</li><li>Feniouk, B. A., Suzuki, T., Yoshida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+subunit+&#949;+in+the+catalysis+and+regulation+of+FOF1-ATP+synthase.">The role of subunit &#949; in the catalysis and regulation of FOF1-ATP synthase.</a> Biochim. Biophys. Acta. 1757, 326-338 (2006).</li><li>Cingolani, G., Duncan, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+ATP+synthase+catalytic+complex+(F1)+from+Escherichia+coli+in+an+autoinhibited+conformation.">Structure of the ATP synthase catalytic complex (F1) from Escherichia coli in an autoinhibited conformation.</a> Nat. Struc. Mol. Biol. 18, 701-707 (2011).</li><li>Mirsaeidi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=After+40+years,+new+medicine+for+combating+TB.">After 40 years, new medicine for combating TB.</a> Int. J. Mycobacteriol. 2, 1-2 (2013).</li><li>Shah, N. B., Hutcheon, M. L., Haarer, B. K., Duncan, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F1-ATPase+of+Escherichia+coli:+The+&#1013;-inhibited+state+forms+after+ATP+hydrolysis,+is+distinct+from+the+ADP-inhibited+state,+and+responds+dynamically+to+catalytic+site+ligands.">F1-ATPase of Escherichia coli: The &#1013;-inhibited state forms after ATP hydrolysis, is distinct from the ADP-inhibited state, and responds dynamically to catalytic site ligands.</a> J. Biol. Chem. 288, 9383-9395 (2013).</li><li>Burgess, T. L., Ross, S. L., Qian, Y. -. x., Brankow, D., Hu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthetic+Processing+of+neu+Differentiation+Factor:+glycosylation,+trafficking,+and+regulated+cleavage+from+the+cell+surface.">Biosynthetic Processing of neu Differentiation Factor: glycosylation, trafficking, and regulated cleavage from the cell surface.</a> J. Biol. Chem. 270, 19188-19196 (1995).</li><li>Loo, T. W., Clarke, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Topology+of+a+Cysteine-less+Mutant+of+Human+P-glycoprotein.">Membrane Topology of a Cysteine-less Mutant of Human P-glycoprotein.</a> J. Biol. Chem. 270, 843-848 (1995).</li><li>Tsao, K. -. L., DeBarbieri, B., Michel, H., Waugh, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+plasmid+expression+vector+for+the+production+of+biotinylated+proteins+by+site-specific,+enzymatic+modification+in+Escherichia+coli.">A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli.</a> Gene. 169, 59-64 (1996).</li><li>Dayne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioLayer+Interferometry+(BLI)+-+How+Does+it+Work?.">BioLayer Interferometry (BLI) - How Does it Work?.</a> Fort&#233;Bio newsletter In Interactions. 5, 8-9 (2012).</li><li>Wartchow, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensor-based+small+molecule+fragment+screening+with+biolayer+interferometry.">Biosensor-based small molecule fragment screening with biolayer interferometry.</a> J. Comput. Aided Mol. Des. 25, 669-676 (2011).</li><li>Abdiche, Y., Malashock, D., Pinkerton, A., Pons, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+kinetics+and+affinities+of+protein+interactions+using+a+parallel+real-time+label-free+biosensor,+the+Octet.">Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet.</a> Anal. Biochem. 377, 209-217 (2008).</li></ol>

作者宣称，他们有没有利益冲突。

对于BLI目前可用的工具允许用于检测生物分子相互作用显著吞吐量和灵活性。各种溶液的样品被分配在一个黑色微量滴定板的孔中，和一组平行的BLI传感器被编程以来回移动孔在板的列之间。将样品通过轨道振荡整个测定搅拌。这里所使用的系统具有8个传感器，并且使用96孔样品盘，但另一个系统采用16个传感器和一个384孔的样品板。因此，一个传感器固定化的生物分子，并在溶液中的结合配偶?...

蛋白质-蛋白质相互作用是重要的，许多生物过程和无标记光学方法，如表面等离子体共振（SPR）已被用于在体外研究的结合和解离动力学1。大多数无标记方法固定在传感器表面1的生物分子，并使用一个光信号，从溶液中检测结合配偶体，因为它与固定的生物分子1相关联。而SPR是一种高度灵敏的方法，很容易产生干扰的变化导致流过传感器2的溶液的折射率。虽然不如SPR，生物层干涉测量（BLI）作为敏感较少受样本结构改变1,3。 BLI使用具有在前端的专有生物相容性涂层光纤生物传感器。这里使用（八位-RED96）系统包含八个分光光度计。白光通过管道输送到某行上移动机械臂探头。光纤传感器ARË拾起探头和转移到含有样品96孔板。一种靶分子被固定在生物传感器表面。然后传感器被移动到包含在溶液中的结合配偶体的孔中。劳保局监察与固定化分子的结合合作伙伴的关联，然后移动传感器解决方案，而约束力的合作伙伴后，监控解离。分子结合到生物传感器表面导致其反射回分光光度仪从内部表面和从传感器和溶液之间的外部接口的光波之间的变化的光学干涉。这些变化中的干扰可以被量化，并用于确定结合和解离，总结在图1所示的动画动力学速率。 我们已申请BLI测量细菌ATP合酶的催化复杂，其ε亚基，它可以自动抑制酶之间的相互作用。 ATP学年nthase是膜嵌入旋转纳米发动机，其催化合成与ATP的水解4。该催化复合物（F 1）可以分离的，因为可以作为一个ATP酶的可溶形式。亚基ε具有两个结构域：N-末端结构域（NTD）是必要的适当组件和所述酶的功能性偶联，但不直接与催化亚基相互作用;的C-末端结构域（CTD）可以通过与相互作用抑制酶多重催化亚基5,6。这ε-介导的调控是针对细菌的ATP合成酶和线粒体同源没有观察到。 ATP合酶已经成为了抗菌药物的目标，如由bedaquiline治疗耐药结核病7近期FDA的批准。因此，靶向药物发现ε的抑菌作用可能产生抗菌素不抑制线粒体ATP合酶。与孤立的催化性复合物（F 1），ε成为一个可分离的亚基。然而，随着ε绑定到F 1，εCTD可经历戏剧性的构象变化，部分地插入到酶的中央空腔，并形成有抑制作用的状态是不太可能直接6,8解离。我们使用BLI测量F 1 /ε结合和解离动力学，间接，审查的催化位点配体对ε的构象变构效应。 在我们的系统中，ε自BLI信号（如SPR）被选择用于固定在传感器表面上是对分子的表面结合的质量敏感。 ε亚基是小（约15 kDa）的相对于所述主F 1复合物（〜347 kDa）的。从而，一个更大的BLI信号将导致选自F 1的结合固定化的ε。为了监测F 1的解离，它可以是非常慢，ε必须被牢固地固定。因此，我们选择了生物素化以及它固定在链亲和素包被的生物传感器。蛋白质可以通过（i）随机修饰表面的赖氨酸9的，用生物素-马来酰亚胺试剂10或（iii）基因中加入特定的生物素受体 ​​肽，它是在酶促生物素化的独特的天然或改造的半胱氨酸（II）的反应是生物素化在标签的蛋白11的体内表达。在我们的系统中，ε是用方法（ⅲ）8生物素化。一旦生物素标记的ε固定在链霉亲和传感器，劳保局可以测量的结合和F 1的解离已耗尽亚基ε（F 1（-ε））的。对于这里描述的实验中，初步试验已经完成，以确定合理的量的生物素化的蛋白质与固定在传感器上。这可以有所不同，这取决于蛋白质的分子量和其结合配偶体，但目标是确定的固定化蛋白质吨最小量帽子（i）提供可接受的信号与噪声的结合动力学与结合配偶体（下面杀敌D）的低浓度和结合动力学与结合配偶体的附近的饱和浓度（ⅱ）最小的失真。此外，生物素化的化学计量可能会有所不同（但要避免&gt; 1摩尔生物素/摩尔蛋白），所以一些初步的分析可能需要为每一批新的生物素化的蛋白质，以确认一致劳保局信号可以固定化过程中实现对链霉亲和涂层传感器。

<table border="0" cellpadding="0" cellspacing="0" width="610">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Octet-RED96</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">30-5048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">A6003-10G</td>
			<td>Fatty Acid free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biosensor/Streptavidin</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">18-5019</td>
			<td>Tray of 96 sensors</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Microtiter plate</td>
			<td style="width:108px;">Greiner Bio-one</td>
			<td style="width:119px;">655209</td>
			<td>Black, Polypropylene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Data Acquisition software</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">Version 6.4</td>
			<td>Newer versions available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Data Analysis software</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">Version 6.4</td>
			<td>Newer versions available</td>
		</tr>
	</tbody>
</table>

bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects

我们描述了使用生物层干涉的研究ε亚基的抑制的相互作用与大肠杆菌 ATP合酶的催化复杂。细菌F型ATP合酶 是一个新的，FDA批准的抗生素对抗耐药结核病的目标。由亚基ε的C-末端结构域理解菌特异性自动抑制ATP合酶可以提供一个新的方法对目标酶对发现的抗菌药物。 ε的C端结构域发生时的有效和无效状态，和催化位点的配体之间的转换酶能影响其中ε的构象主要是一个戏剧性的构象变化。 ε的绑定/解离与催化剂络合物，并间接MEA的检测措施，动力学SURES酶结合ε的移位，并从表面上nondissociable抑制性构象。生物层干涉测量信号不是过于敏感，溶液组合物，因此它也可以被用于监视的催化位点的配位体上的ε的构象变化的变构效应。

实时结合和解离劳保局动力学如图3所示。这个实验是在结合和解离与分析缓冲液进行。该实验开始10分钟基线的步骤，因为这些传感器已被预先润湿仅简要介绍。接着，生物素化的ε被加载在传感器上。 ε中没有检测到发生解离整个从参考曲线（G）因无约束力的合作伙伴加入所看到的所有剩余的步骤。第二个参考传感器（H）为缺乏固定的蛋白质，呈现出结合合作伙伴的低非特异性结?...

Watch this Scientific Journal Video about Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects at JoVE.com

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

这里的协议描述的蛋白质 - 蛋白质相互作用与生物层干涉动力学分析。 F型ATP合成酶，它参与了细胞的能量代谢，可通过在细菌中的ε亚基抑制。我们已经适应生物层干涉研究催化复杂的相互作用与ε的抑制C端结构域。

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 ...

bio-layer-interferometry-for-measuring-kinetics-protein-protein

Research

JoVE Journal

Chemistry

28.7K 次观看.  SUNY Upstate Medical University. 这里的协议描述的蛋白质 - 蛋白质相互作用与生物层干涉动力学分析。 F型ATP合成酶，它参与了细胞的能量代谢，可通过在细菌中的ε亚基抑制。我们已经适应生物层干涉研究催化复杂的相互作用与ε的抑制C端结构域。 

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

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

一种简单快速的协议用荧光测量中性脂肪在藻细胞

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

科研

化学

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

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 ...

Synthesis of Ligand-free CdS Nanoparticles within a Sulfur Copolymer Matrix

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature solvents. These coordinating ligands help solubilize and stabilize the nanoparticles while in solution, and can influence the resulting size and reactivity of the nanoparticles during their formation. Despite the ubiquity of using ligands during synthesis, the presence of aliphatic ligands on the nanoparticle surface can result in a number of problems during the end use of the nanoparticles, necessitating further ligand stripping or ligand exchange procedures. We have developed a way to synthesize cadmium sulfide (CdS) nanoparticles using a unique sulfur copolymer. This sulfur copolymer is primarily composed of elemental sulfur, which is a cheap and abundant material. The sulfur copolymer has the advantages of operating both as a high temperature solvent and as a sulfur source, which can react with a cadmium precursor during nanoparticle synthesis, resulting in the generation of ligand free CdS. During the reaction, only some of the copolymer is consumed to produce CdS, while the rest remains in the polymeric state, thereby producing a nanocomposite material. Once the reaction is finished, the copolymer stabilizes the nanoparticles within a solid polymeric matrix. The copolymer can then be removed before the nanoparticles are used, which produces nanoparticles that do not have organic coordinating ligands. This nascent synthesis technique presents a method to produce metal-sulfide nanoparticles for a wide variety of applications where the presence of organic ligands is not desired.

Herein we present a method to synthesize ligand-free cadmium sulfide (CdS) nanoparticles based on a unique sulfur copolymer. The sulfur copolymer operates as a high temperature solvent and a sulfur source during the nanoparticle synthesis and stabilizes the nanoparticles after the reaction.

a硫磺共聚物基质内的免费配体硫化镉纳米粒子的合成

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature ...

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

定点自旋标记和五聚体配体门控离子通道的EPR光谱研究

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, 1,4-benzene-bis(iminoguanidinium) (BBIG), is demonstrated. The ligand is synthesized as the chloride salt (BBIG-Cl) by in situ imine condensation of terephthalaldehyde with aminoguanidinium chloride in water, followed by crystallization as the sulfate salt (BBIG-SO4). Alternatively, BBIG-Cl is synthesized ex situ in larger scale from ethanol. The sulfate separation ability of the BBIG ligand is demonstrated by selective and quantitative crystallization of sulfate from seawater. The ligand can be recycled by neutralization of BBIG-SO4 with aqueous NaOH and crystallization of the neutral bis-iminoguanidine, which can be converted back into BBIG-Cl with aqueous HCl and reused in another separation cycle. Finally, 35S-labeled sulfate and &#946; liquid scintillation counting are employed for monitoring the sulfate concentration in solution. Overall, this protocol will instruct the user in the necessary skills to synthesize a ligand, employ it in the selective crystallization of sulfate from aqueous solutions, and quantify the separation efficiency.

A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented.

分离硫酸选择性结晶用双 -  iminoguanidinium配体

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Accessing Valuable Ligand Supports for Transition Metals: A Modified, Intermediate Scale Preparation of 1,2,3,4,5-Pentamethylcyclopentadiene

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that derive from initial 2-bromo-2-butene lithiation followed by acid mediated dienol cyclization. The revised synthesis and purification of the ligand avoids the use of mechanical stirring while still permitting access to significant quantities (39 g) of Cp*H in good yield (58%). The procedure offers other additional benefits, including a more controlled quench of excess lithium during the production of the intermediate heptadienols and a simplified isolation of Cp*H of sufficient purity for metallation with transition metals. The ligand was subsequently used to synthesize [Cp*MCl2]2 complexes of both iridium and ruthenium to demonstrate the utility of the Cp*H prepared and purified by our method. The procedure outlined herein affords substantial quantities of a ubiquitous ancillary ligand support used in organometallic chemistry while minimizing the need for specialized laboratory equipment, thus providing a simpler and more accessible entry point into the chemistry of 1,2,3,4,5-pentamethylcyclopentadiene.

Reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented. The revised protocol for the synthesis and purification of the ligand minimizes the need for specialized laboratory equipment while simplifying reaction workups and product purification. Use of Cp*H in the synthesis of [Cp*MCl2]2 complexes (M = Ru, Ir) is also described.

访问有价值的配体为支持过渡金属：1,2,3,4,5-五甲基环戊二烯的一种改进，中级规模制备

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

配体介导成核与钯金属纳米粒子的生长

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.