Name: multiplexed single-molecule force proteolysis measurements using magnetic tweezers
Uploaded: 2012-07-25T11:00:00
Description: Watch this Scientific Journal Video about Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers at JoVE.com

这项工作是支持的巴勒斯惠康在科学界面（ARD），国立卫生研究院通过国立卫生研究院主任的新的创新奖励计划1-DP2-OD007078（ARD）的威廉·鲍斯小斯坦福大学研究生奖学金（ASA的事业奖），斯坦福大学心血管病研究所年轻的博士前奖学金（JC），。作者感谢出借显微镜设备詹姆斯Spudich。

1。流动电池的制备<ol><li>盖玻片（＃1.5，22x22毫米和22x40毫米，厂商VWR）使用超声波清洗。 <ol><li>添加盖玻片盖玻片和装修在sonicator（见步骤2）的一个小玻璃容器。 </li><li>填补与异丙醇的容器和超声在20分钟的沐浴sonicator。 </li><li>丢弃异丙醇和MilliQ Barnsted设备或类似设备由去离子水冲洗量，冲洗盖玻片。装满水的容器中，超声20分钟。 </li><li>超声波处理后，干燥过滤，无粉尘的空气流中的盖玻?...

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本协议描述了一个经典的单分子技术的新用途。磁性镊子允许介质在成本效益的方式，以高通量单分子检测。然而，像所有的实验技术，也有挑战和潜在的隐患。 磁性镊子的限制磁光阱相比，一吨设备的空间和时间分辨率低。此外，这里描述的简单吨，产生的力是30 PN或以下，明显比AFM实验中经常访问的力量。这种限制可以是一种美德：吨是适合申请子-PN?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>微盖玻璃＃1.5（22X22） </td><td>厂商VWR </td><td> 48366-067 </td></tr><tr><td>微盖玻璃＃1.5（22x40） </td><td>厂商VWR </td><td> 48393-048 </td></tr><tr><td>噬菌体DNA </td><td> Invitrogen公司</td><td> 25250-010 </td></tr><tr><td> T4 DNA连接酶</td><td> Invitrogen公司</td><td> 15224-041 </td></tr><tr><td>单片机Ultracel YM-100 </td><td> Millipore公司</td><td> 42413 </td></tr><tr><td>抗地高辛</td><td>罗氏诊断</td><td> 11-333-089-001 </td></tr><tr><td>吐温20 </td><td>西格玛</td><td> P9416-100ML </td></tr&gt; <tr><td>抗-myc基因抗体</td><td> Invitrogen公司</td><td> 46-0603 </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> B4287-5G </td></tr><tr><td>磁珠的M-280链霉</td><td> Invitrogen公司</td><td> 658.01D </td></tr><tr><td>磁珠MyOne T1的链霉</td><td> Invitrogen公司</td><td> 658.01D </td></tr><tr><td>醋酸P-Aminophenylmercuric </td><td> Calbiochem公司</td><td> 164610 </td></tr><tr><td>生物素 - 马来酰亚胺</td><td> Sigma Aldrich公司</td><td> B1267 </td></tr><tr><td>生物素标记的寡核苷酸</td><td>内幕交易审裁处的DNA </td><td>定制合成</td></tr><tr><td>地高辛标记寡核苷酸</td><td>内幕交易审裁处的DNA </td><td>定制合成</td></tr><tr><td>胶原肽基因</td><td> DNA的2.0 </td><td>定制合成</td></tr><tr><td>中MMP-1基因</td><td>哈佛质粒数据库</td><td></td></tr><tr><td> Z-翻译</td><td> thorlabs </td><td> MTS50 </td></tr><tr><td>伺服控制器的翻译</td><td> thorlabs </td><td> TDC001 </td></tr></tbody></table>

multiplexed single-molecule force proteolysis measurements using magnetic tweezers

机械力的生成和检测是一个无处不在的细胞生理学方面，直接关系到癌转移1，动脉粥样硬化和伤口愈合3。在这些例子中，每个细胞都在他们的周围施加力量，同时酶解改造外基质（ECM）。流脑的力量的作用也因此成为一个面积相当大的兴趣，由于其可能的生物学和医学的重要性4-7。 如光阱，原子力显微镜，磁镊子10,11单分子技术使研究人员能够探测功能的酶在分子水平上对个别蛋白质的力量发挥。这些技术，磁镊子（吨），其低成本和高吞吐量显着。吨在〜1-100 PN的范围施加的力量，可以提供毫秒级的时间分辨率，12在单分子水平研究酶机制很好地匹配的素质。在这里，我们报告一个高度并行吨的实验研究单个蛋白质分子的蛋白质的影响力。我们提出具体的例子，由基质金属蛋白酶1（MMP-1）的三聚体胶原蛋白肽的蛋白质，但是，此法可以很容易地适应学习其他基板和蛋白酶。

Watch this Scientific Journal Video about Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers at JoVE.com

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

在这篇文章中，我们描述了利用磁性镊子，在一个高度并行的方式在单分子水平研究的酶水解的影响力。

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 ...

Research

JoVE Journal

Bioengineering

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

伸展的DNA短序列恒力轴向光镊

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

科研

生物工程

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

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 ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Magnetic Tweezers for the Measurement of Twist and Torque

磁性镊子的扭曲和扭矩的测量

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

快速扫描电子顺磁共振打开新的途径成像生理重要参数<em&gt;在体内</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

构建用于扩展视场单分子成像的高倾角扫描磁贴显微镜的指南

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

使用荧光显微镜在剪切流下描述单分子构象变化

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...