Name: flexural rigidity measurements of biopolymers using gliding assays
Uploaded: 2012-11-09T13:00:00
Description: Watch this Scientific Journal Video about Flexural Rigidity Measurements of Biopolymers Using Gliding Assays at JoVE.com

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...

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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?...

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?...

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 ...

Research

JoVE Journal

Bioengineering

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

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

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

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

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

珠聚集测定假定的细胞粘附分子的表征

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Molecular Entanglement and Electrospinnability of Biopolymers

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

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

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

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 ...