我们协助准备图1和安娜·拉特利夫证明了协议的感谢梅丽莎克洛克。这项工作是由研究公司的科技进步。

1。微管滑动含量库存解决方案提前准备的滑翔试验。 <ol><li>聚合0.5毫克明亮的有机荧光22标记的微管人烟稀少。目标标签的浓度为1每微米的微管，或荧光标记密度约每1500个微管蛋白二聚体的荧光。储存在室温温度，光照，保护与铝，铝箔，长达两个星期。 </li><li>纯化生物素驱动蛋白21在约1μM。储存在-80℃，在5微升的等分试样用于在个别实验。 </li><li>准备的测定缓冲液（A，B）的50mM imidiazole，50mM的氯化钾（KCl），4 mM氯化镁（MgCl 2）中，2mM的乙二醇四乙酸（EGTA），pH值6.7。无菌过滤器，并储存于4°C。 </li><li>将生物素化的牛血清白蛋白（生物素-BSA溶解）2毫克/毫升在AB。与0.2微米针头式过滤器过滤。储存在-80℃，在长时间的100μl等分试样，在4℃下长达一个月。 </li><li>将链霉亲和素（SA）溶解至10毫克/毫升在AB。与0.2微米针头式过滤器过滤。储存在-80℃下在20微升等份长时间，在4℃下，最多两个星期。 </li><li>将α-酪蛋白溶解至5毫克/毫升，在AB。与0.2微米针头式过滤器过滤。储存在-80℃，在长时间的100μl等分试样，在4℃下进行最多两个星期。 </li><li>在200mM的二硫苏糖醇（DTT）在去离子水中溶解。在100微升等份，储存在-20°C解冻后8小时内使用。可以冷冻。 </li><li> 4mM的HPLC级的二甲基亚砜（DMSO）中溶解紫杉醇（PT）。商店在10微升等份，在-80°C长期，-20°C短期。解冻后8小时内使用。可以冷冻。 </li><li>溶解在去离子水中的葡萄糖，在120毫克/毫升。贮存于-20℃下在100μl等份可以冷冻。 </li><li>派息解决的三磷酸腺苷（ATP）在去离子水中，在150mM，pH为7.0。在-80°C存放在5微升等份，解冻后8小时内使用。 </li><li>准备好100倍除氧股票25到600μl总分析缓冲液溶解10,000个单位的葡萄糖氧化酶和过氧化氢 ​​酶156,000单位。短暂离心，离心沉淀固体;上清液用0.2微米针头式过滤器的过滤器。储存在-80℃，于10μl等分长时间，在4℃下最多为一个星期。 </li></ol> 2。微管滑翔含量，即日溶液的制备准备在2.1-2.6的解决方案在实验当天。通过实践，解决方案，准备在2.2-2.6流动池清洗。除非另有说明，在冰上保持库存中的解决方案。 <ol><li>准备AB，1毫升测定缓冲液中，用10微升的DTT股票（AB用2mM DTT）。在室温下保存。 </li><li>准备生物BSA缓冲液，40亩; l的生物素-BSA的库存和AB的1:1混合物中。在室温下保存。 </li><li>准备BSA缓冲液中，用5.5微升BSA（1毫克/毫升BSA在AB）645微升AB混合。在室温下保存。 </li><li>准备SA的缓冲液，用3微升的链霉抗生物素蛋白的股票（0.5毫克/毫升链霉抗生物素蛋白在AB）将57微升AB混合。在室温下保存。 </li><li>制备α-酪蛋白的缓冲液，混合200μl的BSA缓冲液中，加入50μl的酪蛋白的库存中，和0.8微升15μMATP（1毫克/毫升α-酪蛋白，0.8毫克/毫升BSA，50 nM的ATP在AB）。在室温下保存。 </li><li>准备荧光防漂白剂缓冲，将95微升α-酪蛋白缓冲，2.5微升葡萄糖股票，1微升100倍的氧清除组合，1微升紫杉醇股票，1微升2 - 巯基乙醇（βME）混合。添加βME下通风柜。使用荧光漂白剂抗缓冲区内1小时的准备。的注意 :βME是剧毒的，味道很难闻。请务必只在通风橱下打开。在商店瓶没有光亮;的光降解βME和能力，以减少漂白。 </li></ol> 3。微管滑翔检测<ol><li>建设24×60毫米盖玻片和22 x 22毫米盖玻片四流道之间采用真空油脂，通过了一套吸头注射器挤出，作为间隔。每个流道应该是约10微升量（规模相应的后续卷）。 </li><li>洗到每一个车道的生物BSA缓冲液10微升。孵育5分钟以允许BSA的涂覆的玻璃表面。 </li><li>每个泳道洗净用15μlBSA缓冲液中，以除去游离的生物素-BSA的3倍，同时继续阻止的滑动表面。使用一个的Kimwipe或滤纸，删除流室对面的缓冲区， 请确保不要让气泡通过流室 。 </li><li> SA-15μL缓冲液洗到每个车道。等待10分钟，或最多2个小时。链霉亲和素将绑定到表面结合生物素-BSA。 </li><li>每个泳道洗净三次用15μlBSA缓冲液中，以除去游离的SA，同时继续以阻止滑动表面。 </li><li>每个泳道洗净用15μlα-酪蛋白的缓冲。 α-酪蛋白，可以进一步阻止非特异性结合的动蛋白，微管的玻璃。 </li><li>稀释驱动蛋白10nM的α-酪蛋白的缓冲液中，并用15μl此驱动蛋白 - α-酪蛋白溶液洗每个泳道。等待15分钟（或长达一个小时）的生物素化的驱动蛋白特异性结合的表面结合的链霉抗生物素蛋白。将继续免费绑定微管的驱动蛋白马达域。 </li><li>稀释紫杉醇α-酪蛋白缓冲液在室温（40μM），并用15微升该溶液洗每个泳道洗出自由驱动蛋白和预加载每个流室与紫杉醇溶液，以防止从解聚的微管。冷解聚微管，所以一定要确保这些步骤发生在室温下用房间temperatu重新解决方案。 </li><li>稀释荧光标记微管1:100-1:1,000在1 mM ATP荧光漂白剂抗缓冲。 15μL洗成一个车道，并在30分钟内观察。 </li></ol> 4。数据采集<ol><li>用荧光显微镜观察微管滑翔;，显微镜需要能够解决单个荧光基团，如商业或家庭构建TIRF设置的26（ 图2）。 应当由驱动蛋白马达推进微管，在该衬底上在约0.5微米/秒，这取决于温度。如果该字段的视图的显微镜是50微米，单个微管应该是可见的约100秒。设置单荧光基团，使光照强度不超过100秒的光漂白剂更迅速。如果使用激光激发，约3-5毫瓦的功率设置是合适的。 </li><li>收藏报错序列的图像进行分析。 600图像5赫兹（共2分钟）效果很好。使用序列足够长的时间，微管遍历整个视场的。 </li></ol> 5。数据分析一个IDL的常规， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50117/50117IDL_Routines.zip" target="_blank">get_lp.pro</a> ，连接。此例程返回一个持久性的基础上所有微管滑翔在一个给定的图像序列的长度值。运行这个程序对每一个图像序列，修改强度参数，根据您的特定显微镜设置，或执行以下操作: <ol><li>跟踪每一个荧光团连接到微管的荧光轨迹。 </li><li>合并所有的轨迹的一个给定的微管成一个整体的微管轨迹上的荧光基团，重复的图像序列中的每个微管（ 图3）。 </li><li>计算每个点沿轨迹的切线角度（ 图4）和计算&lt;cosθs的&gt;在方程1，平均每对分离通过的路径长度 s（ 图5）的点的角度差的余弦。找到持久的长度为一个单一的微管或微管组拟合&lt;cosθS&gt;式。 1，单独的数据点中的适合由数量的独立的cosθs的值被用来计算平均加权。 </li></ol>

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作者宣称，他们没有竞争的金融利益。

持久性长度测量是个别的生物聚合物的机械性能良好的表征。在这篇文章中，我们已经描述的方法测量微管长度的持久性。如在引言中指出的，这种方法很容易扩展到检查微管的机械性能在各种条件下，简单地通过不同的试剂，温度或粘度的滑动试验中，3.9，在最后步骤中，或通过聚合微管，步骤1.1，在不同的条件下。 技术本身是迅速执行。一旦股票方案已经提前准备的时间（第1步），滑翔检测实验可以在2-3小时，其中包括四个变化的条件下，在一台显微镜幻灯片（四车道的步骤3.1）。许多微管可以检查每个实验的典型实验，在我们的实验室检查100微管的条件（约10图像测序ES，每个通道，每个序列有10微管）。分析大型数据集同样是相当快的。通过练习，10个图像序列进行分析，持效期长的一个下午或一个现代PC。在分析的限速步骤是跟踪单个荧光基团;新的跟踪方法保证速度大幅增加27。 因为该方法涉及跟踪单个荧光团连接到一个给定的微管，跟踪精度只限于由光子收集，并可以在纳米22，25的规模。通过包括数据从许多荧光基团，进一步提高精度。潜在的，甚至更高的精度可以达到非常明亮的荧光物体，如量子点连接，微管。 虽然个人轨迹的精度是相当不错的，持久性的不确定性乐用这种方法测得的ngths（在一个单一的微管规模的±50％）仍然是显着的。持效期长测量精度的限制，关键是统计平均COSθS。作为分， 秒 ，增加的对之间的轮廓长度，θs的独立测量的数目减少为 L / s时， 其中 ，L是微管轨迹长度的研究。增加微管轨道的长度允许的持久长度测量的精度。 这种方法的第二个限制是，生物素化的动蛋白被用于特定的附件驱动蛋白显微镜玻片上的要求。生物素标记的驱动蛋白纯化（从大肠杆菌在我们的例子中），并不能简单地购买现成的。但是，修改后的滑翔试验使用unbiotinylated亲属ESIN可以使用28;驱动蛋白重链蛋白质，可能会对适合这种技术从细胞骨架（KR01）是市售的。修改的滑翔试验，使用此备用动蛋白不会影响微管的抗弯刚度以任何方式，从而使重组动蛋白组没有使用此方法。

细胞骨架，大多数真核细胞中发现的生物聚合物网络，起到了重要作用的细胞组织，细胞内运输，细胞力学。细胞骨架的生物聚合物（主要是肌动蛋白和微管）的机械特性，发挥了显着的作用，作为一个整体12在确定的细胞的机械特性。由于整个细胞力学可以表征健康和患病细胞13,14和涉及在蜂窝蠕动15，底层细胞骨架成分的机械性能，在过去的二十年里1是一个活跃的研究领域。 的生物聚合物的弹性（或刚性），其特征在于由持久长度，长度的聚合物，该聚合物由约1弧度弯曲下在环境温度下的热波动。已经开发了许多技术测量持续长度16，用于examp乐活跃的技术，包括弯曲的聚合物水动力流，光学陷阱，或电场4,17,18，和被动的技术，测量波动的自由聚合物溶液中5,6。的活性的测量，但是，需要专门的设置，以实现的微米尺度上的已知的力量，和自由波动测量可以是具有挑战性由于扩散满分使用显微镜的焦点的平面，。 在这篇文章中，我们描述了一种互补的，被动的，技术来衡量的持效期长的微管，细胞骨架聚合物。该技术涉及滑动检测，确保聚合物始终保持在焦平面19。此外，它涉及到跟踪单个荧光基团连接永久的聚合物的利益，使特定的位置沿聚合物很好的特点。 一个卡通的方法显示在FIGURE 1。动蛋白专门向"+"端的微管移动，因此推动单向微管的滑翔试验。超出附加的最后驱动蛋白，微管的前端是自由浮动，根据周围的溶液的热势力。由于微管向前推进，最终波动，直到绑定到一个新的驱动蛋白分子沿玻璃幻灯片冻结在一个给定的波动。由于动蛋白非常重视微管，强烈的约束，微管的前端走的一条路。因此，微管轨迹冻成的统计波动是相同的微管11的自由端的统计波动，并因此可以被用来计算至20所述持续长度<img alt="式（1）" fo:content-width="1in" fo:src="/files/ftp_upload/50117/50117eq1highres.jpg" srC =的"/ files/ftp_upload/50117/50117eq1.jpg"/&gt; 其中 l p是持久性的微管的长度，θs是分离的轮廓长度 s的轨迹的切线之间的夹角，&lt;&gt;表示所有对隔开的轮廓长度 s的位置的平均值。 滑翔试验本身采用动蛋白生物素标记的特异性结合的载玻片上通过链霉亲和素生物素联动的双螺旋21。此附件可确保电机领域是自由结合，推动微管。为了顺应微管的轨迹，人烟稀少标记微管与有机荧光22,23 -标签必须足够稀疏，使用单分子荧光显微镜分辨的单荧光团。单荧光团跟踪使用IDL编写的图像分析程序。每个荧光团的轨迹绑定到一个给定的麦克风rotubule被组合成一个复合的微管轨迹自动24。沿一轨迹的每个点的切线的角度θ计算;从这些切线角度&lt;COSθS&gt;的值计算为每个轮廓长度 s。最后，这些数据是符合式。 1，以便对于一个给定的微管中，提取一个持久长度或许多在同一滑翔测定的微管。 该方法是健壮的，足够的工作与在各种各样的条件下（与不同的稳定剂或其它小分子的微管结合，与绑定微管相关蛋白（MAPs），或与各种粘稠溶液）制备的微管。在我们的实验室中，该技术已被用于表征作为的函数的长度沿不同的稳定剂的微管和微管与微管长度的持久性。主要的限制是微管必须小号支持动蛋白的活力。由于动蛋白是一个强大的电机酶，这是一个相当宽松的限制。通过更换微管与肌动蛋白和驱动蛋白与肌球蛋白家族酶，持效期长的肌动蛋白可以使用相同的技术。

<table border="1"&gt;<tbody&gt;<tr&gt;<td</td&gt;<td</td&gt;<td</td&gt;<td<strong&gt;试剂</strong</td&gt;</tr&gt;<tr&gt;<td&gt;咪唑</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; I2399</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;氯化钾</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; P9541</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;氯化镁</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; M8266</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; EGTA</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; E3889</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; BSA</td&gt;<td&gt; Calbiochem公司</td&gt;<td&gt; 126615</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;生物素标记的牛血清白蛋白</td&gt;<td&gt;赛默飞世尔科技</td&gt;<td&gt; 29130</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;α-酪蛋白</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; C6780</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;链霉亲和素</td&gt;<td&gt;赛默飞世尔科技</td&gt;<td&gt; 21125</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;二硫苏糖醇</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; D0632</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;紫杉醇</td&gt;<td&gt; LC实验室</td&gt;<td&gt; P-9600</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td葡萄糖氧化酶</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; G2133</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;过氧化氢酶</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; C100</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;葡萄糖</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; G8270</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; ATP</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; A2383</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 2 - 巯基乙醇</td&gt;<td&gt; Sigma-Aldrich公司</td&gt;<td&gt; M3148</td&gt;<td&gt;毒性。购买量小。</td&gt;</tr&gt;<tr&gt;<td&gt; 24X60毫米编号1 1/2玻璃盖</td&gt;<td&gt; VWR</td&gt;<td&gt; 48393-252</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 22X22毫米1号的封面玻璃</td&gt;<td&gt;金印</td&gt;<td&gt; 3306</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;高真空润滑脂</td&gt;<td&gt;陶氏 - 康宁</td&gt;<td&gt; NA</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td</td&gt;<td</td&gt;<td</td&gt;<td<strong&gt;设备</strong&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; TIRF显微镜</td&gt;<td&gt;许多</td&gt;<td&gt; NA</td&gt;<tdTIRF显微镜使用这种方法是自制的。</td&gt;</tr&gt;<tr&gt;<tdIDL（软件）</td&gt;<td&gt; Exelis</td&gt;<td&gt; NA</td&gt;<td可以代替MATLAB，ImageJ的，或其他图像分析软件。</td&gt;</tr&gt;</tbody&gt;</table

flexural rigidity measurements of biopolymers using gliding assays

微管是细胞骨架的聚合物在细胞分裂中发挥作用，细胞力学，细胞内的运输。这些函数中的每一个都需要微管僵硬，不够直，跨越了显着的部分的细胞直径。其结果是，微管持续长度，刚度的措施，已积极研究在过去的二十年1。然而，开放式的问题依然存在:短微管的10-50倍比长的微管2-4僵硬，甚至长的微管测量的持久性不同的长度，命令5-9级。 在这里，我们提出了一种方法来衡量微持效期长。该方法是基于对动蛋白驱动的微管滑翔测定10。通过结合稀疏的荧光标记的个人微管的单粒子跟踪个人的微管连接的荧光基团，glidin克轨迹跟踪单微管的纳米级精度。的持久性的轨迹长度是相同的持久性的微管长度的条件下，使用了11。一个自动跟踪例程被用来创建从荧光团连接到个别微管的微管的轨迹，这个轨迹的持续长度计算使用在IDL编写的例程。 此技术是迅速实现的，并且能够在一天的实验测量的持续长度为100微管。该方法可以扩展到在各种条件下，包括沿微管长度的函数的持续长度为测量持久长度。此外，所使用的分析例程可以被扩展到基于肌球蛋白的作用滑翔检测，测量的持久性，以及肌动蛋白丝长度。

快照滑翔试验， 如图2所示。一个很好的微管密度为1-10微管，每场来看，大幅度增加将导致错误跟踪，微管相互交叉。甲地积的11个微管从在图2中的滑动检测的轨迹在图3所示。典型的轨迹是10到30微米长，有的轨迹有差距的其中一个微管穿过另一。这些轨迹从分析中可能会被丢弃。 一个单一的，长的微管的轨迹示出在图4A中沿轨迹在两个位置与例如切线夹角。由一个固定的距离分隔的切线夹角之间的差别有关的持续长度，沿着微管轨迹的位置作为一个功能是在图4B所示的切线的角度。 角正切数据从许多微管轨迹相结合来计算一个单独的持久长度图5示出了可用于所有在一个给定的实验的微管。&lt;cosθs的 &gt;与轮廓长度 s滑翔检测图2的曲线图。在大的轮廓长度值，很少的cosθ值计量，因此平均高度可变的。加权拟合符合短期小号 ，高精度的数据更加紧密地渴望着，低精度的数据。 持久性从这些11的轨迹的长度，500±40微米（平均值±标准误差），该值是相对短的微管的长度为2的持久性的代表。类似的实验，300至1000微米的范围内的持久性长度。 故障排除如果微管是完全不存在的，增量酶的浓度的微管在步骤3.9到0.5毫克/毫升。如果微管人仍下落不明，重新聚合微管。确保紫杉醇是目前每个解决方案中稀释成微管，否则微管解聚。 如果微管的解决方案，但不具约束力的表面自由扩散，增加动蛋白的浓度在3.7步骤和使用AMP-PNP，而不是ATP，以确保动蛋白结合微管不可逆的。如果微管还没有绑定，使新的链霉亲和素股票和缓冲液中，通过新的生物素-BSA缓冲。最后，净化新的生物素化的驱动蛋白。 如果微管结合，但不动，更换新鲜的ATP ATP原液。 如果荧光基团的光漂白速度太快，降低光照强度。如果荧光基团的光漂白速度太快，更换氧清除系统的新货。 如果fluoropho水库光线太暗，增加光照强度。 <img src="/files/ftp_upload/50117/50117fig1.jpg" alt="图1" /> 图1。卡通的微滑翔试验。动蛋白的酶特异性结合的生物素 - 链霉亲和素联动盖玻片。人烟稀少的有机荧光标记微管。加入ATP后，微管推由驱动蛋白电机。的领先的微管的自由端，由于在溶液中的热力波动，这些波动被用来计算微管持续长度。这些实验中所探测的微管的长度规模的自由端的长度。 <img src="/files/ftp_upload/50117/50117fig2.jpg" alt="图2" /> 图2:典型的滑翔试验，通过全内反射荧光显微镜显微镜快照。微管<html> bules稀疏的装饰与单个荧光基团。比例尺为5μm。 <img src="/files/ftp_upload/50117/50117fig3.jpg" alt="图3" /> 在图2中所示的图3，微管从所述图像序列的轨迹。每个微管轨迹结合许多单个荧光轨迹（约10平均），和已减薄到100nm的每一个点。 <img src="/files/ftp_upload/50117/50117fig4.jpg" alt="图4" /> 图4计算的轨迹的切线的角度。 （A）的轨迹的一个微管中，示例性示出的切线的角度（θ）。 （B）的切线角度沿着微管轨迹的位置作为一个功能。这些数据被用于计算方程中使用的平均角度。 1。 AYS"&gt; <img src="/files/ftp_upload/50117/50117fig5.jpg" alt="图5" /> 图5。从切线角度计算持效期长。 （ 点 ）为图3中所示的11个轨迹剧情&lt;cosθs的 &gt;的轮廓长度 s。 （ 实线 ）调整到式。 1。这组微管，持效期长为500±40微米。对于长的轮廓的长度（10微米或以上），数据是高度可​​变的，由于有限的统计数据。

Watch this Scientific Journal Video about Flexural Rigidity Measurements of Biopolymers Using Gliding Assays at JoVE.com

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

持效期长或生物大分子的抗弯刚度的方法来衡量。该方法使用一个动蛋白驱动的微管的滑动测定试验方法来测定的持久性个别微管长度，并是适应actin为基础的滑翔检测。

使用滑翔分析生物大分子的抗弯刚度测量

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

flexural-rigidity-measurements-of-biopolymers-using-gliding-assays

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.7K 次观看.  Lawrence University. 持效期长或生物大分子的抗弯刚度的方法来衡量。该方法使用一个动蛋白驱动的微管的滑动测定试验方法来测定的持久性个别微管长度，并是适应actin为基础的滑翔检测。

视频: Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

开发自定义的中国仓鼠卵巢宿主细胞蛋白检测使用声膜微粒子技术

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

科研

生物工程

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

微观粒子图像测速技术，微血液流动的速度剖面测量

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

细胞外基质刚性三维肿瘤模型的纵向测量用粒子跟踪Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

珠聚集测定假定的细胞粘附分子的表征

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

分子纠缠与生物大分子的Electrospinnability

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

对干燥空气-LC接口Megamolecular生物大分子的自我整合方法

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

实验室规模搅拌生物反应器的功率输入测量

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

测量海洋海绵针力学性能的毫米尺度弯曲试验系统

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.