警队复用单分子蛋白水解测量磁性镊子
Summary
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。
Abstract
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。
Protocol
1。流动电池的制备盖玻片（＃1.5，22×22毫米和22×40毫米，厂商VWR）使用超声波清洗。 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。 填补与异丙醇的容器和超声在20分钟的沐浴sonicator。 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。 超声波处理后，干燥过滤，无粉尘的空气流中的盖玻?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of Reagent | Company | Catalogue Number |
| Micro Cover Glass #1.5 (22×22) | VWR | 48366-067 |
| Micro Cover Glass #1.5 (22×40) | VWR | 48393-048 |
| Lambda DNA | Invitrogen | 25250-010 |
| T4 DNA Ligase | Invitrogen | 15224-041 |
| Microcon Ultracel YM-100 | Millipore | 42413 |
| Anti-Digoxigenin | Roche Diagnostics | 11-333-089-001 |
| Tween 20 | Sigma | P9416-100ML |
| Anti-myc Antibody | Invitrogen | 46-0603 |
| Bovine Serum Albumin | Sigma | B4287-5G |
| Dynabeads M-280 Streptavidin | Invitrogen | 658.01D |
| Dynabeads MyOne T1 Streptavidin | Invitrogen | 658.01D |
| p-Aminophenylmercuric Acetate | Calbiochem | 164610 |
| Biotin-Maleimide | Sigma Aldrich | B1267 |
| Biotin labeled oligo | IDT DNA | Custom synthesis |
| Digoxigenin labeled oligo | IDT DNA | Custom synthesis |
| Collagen peptide gene | DNA 2.0 | Custom synthesis |
| MMP-1 cDNA | Harvard Plasmid Database | |
| z-translator | Thorlabs | MTS50 |
| Servo controller for translator | Thorlabs | TDC001 |
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Tags
MultiplexedSingle-moleculeForceProteolysisMeasurementsMagnetic TweezersGenerationDetectionMechanical ForcesCell PhysiologyCancer MetastasisAtherogenesisWound HealingExtracellular Matrix (ECM)Enzymatically RemodelForces On ECMBiological ImportanceMedical ImportanceSingle Molecule TechniquesOptical TrappingAtomic Force MicroscopyMagnetic Tweezers (MT)Low CostHigh ThroughputPN (piconewton)Millisecond Temporal ResolutionEnzyme MechanismHighly Parallelizable AssayTrimeric Collagen PeptideMatrix Metalloproteinase 1 (MMP-1)Substrates And Proteases
Cite This Article
Adhikari, A. S., Chai, J., Dunn, A. R. Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers. J. Vis. Exp. (65), e3520, doi:10.3791/3520 (2012).