Name: cargo loading onto kinesin powered molecular shuttles
Uploaded: 2010-11-03T17:00:00
Description: Watch this Scientific Journal Video about Cargo Loading onto Kinesin Powered Molecular Shuttles at JoVE.com

我们负债累累乔纳森豪集团开发的基本协议，后来我们适应一个滑翔运动的检测，其。来自美国国家科学基金会资助DMR0645023的财务支持表示感谢。

1）缓冲液和试剂这些解决方案应提前做好准备，并存储在方便大小等分。等份应包含足够的解决方案一个典型的实验和新鲜等份应为每一个蠕动检测。也提到了以下协议的储存条件和典型等份大小。 1。 BRB80缓冲区，（80毫米的管道，1毫米氯化镁2，1毫米EGTA（DD）在去离子蒸馏水水，由氢氧化钾调整pH至6.9） <ul><li> 100毫升的0.5 M EGTA股市日水的解?...

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	<li>Agarwal, A., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+motors+at+the+intersection+of+nanotechnology+and+polymer+science.">Biomolecular motors at the intersection of nanotechnology and polymer science.</a> Progress in Polymer Science. 35 (1-2), 252-252 (2010).</li><li>Howard, J., Hunt, A. J., Baek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+microtubule+movement+driven+by+single+kinesin+molecules.">Assay of microtubule movement driven by single kinesin molecules.</a> Methods Cell Biol. 39, 137-137 (1993).</li><li>Agarwal, A., Katira, P., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond+curing+time+of+a+molecular+adhesive+causes+velocity-dependent+cargo-loading+of+molecular+shuttles.">Millisecond curing time of a molecular adhesive causes velocity-dependent cargo-loading of molecular shuttles.</a> Nano Lett. 9 (3), 1170-1170 (2009).</li><li>Diez, S., Reuther, C., Dinu, C., Seidel, R., Mertig, M., Pompe, W., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+and+Transporting+DNA+Molecules+Using+Motor+Proteins.">Stretching and Transporting DNA Molecules Using Motor Proteins.</a> Nano Lett. 3 (9), 1251-1251 (2003).</li><li>Bachand, G. D., Rivera, S. B., Boal, A. K., Gaudioso, J., Liu, J., Bunker, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+transport+of+nanocrystal+CdSe+quantum+dot+nanocomposites+using+microtubules+and+kinesin+motor+proteins.">Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins.</a> Nano Lett. 4 (5), 817-817 (2004).</li><li>Coy, D. L., Wagenbach, M., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+takes+one+8-nm+step+for+each+ATP+that+it+hydrolyzes.">Kinesin takes one 8-nm step for each ATP that it hydrolyzes.</a> J. Biol. Chem. 274 (6), 3667-3667 (1999).</li><li>Katira, P., Agarwal, A., Fischer, T., Chen, H. -. Y., Jiang, X., Lahann, J., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+performance+of+protein-resisting+surfaces+at+ultra-low+protein+coverages+using+kinesin+motor+proteins+as+probes.">Quantifying the performance of protein-resisting surfaces at ultra-low protein coverages using kinesin motor proteins as probes.</a> Advanced Materials. 19, 3171-3171 (2007).</li><li>Vigers, G. P. A., Coue, M., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Microtubules+Break+Up+Under+Illumination.">Fluorescent Microtubules Break Up Under Illumination.</a> J. Cell Biol. 107, 1011-1011 (1988).</li><li>Brunner, C., Hess, H., Ernst, K. -. H., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifetime+of+biomolecules+in+hybrid+nanodevices.">Lifetime of biomolecules in hybrid nanodevices.</a> Nanotechnology. 15 (10), S540-S540 (2004).</li></ol>

稍作修改，该协议已经被成功地用于各种群体组装动蛋白，微管为基础的蠕动检测。在最终蠕动解决方案的10毫米数码地面电视服务可以被替换为0.5％β-巯基乙醇。标准解决方案（BRB80AF，KIN20和MT1000）2个多小时，不应该使用。含有紫杉醇，尤其是微管的任何解决方案，不应该被放置在冰上。过度暴露于紫外线激发光损伤的功能部件，结果：流动池，微管和驱动蛋白 8这种效应更为明显，如...

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<td>Adenosine-5&#x2019;-triphosphate (ATP)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1049</td>
<td>&nbsp;</td>
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<td>Biotin tubulin</td>
<td>&nbsp;</td>
<td>Cytoskeleton Inc.</td>
<td>T333</td>
<td>&nbsp;</td>
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<td>Casein</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-0376</td>
<td>&nbsp;</td>
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<td>Catalase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-9322</td>
<td>&nbsp;</td>
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<td>D-(+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7528</td>
<td>&nbsp;</td>
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<td>Dimethylsulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D-8779</td>
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<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#x2032;,N&#x2032;-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E-4378</td>
<td>&nbsp;</td>
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<td>FluoSpheres Biotinylated microspheres, 40 nm, yellow-green fluorescent (505/515)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F-8766</td>
<td>&nbsp;</td>
<tr>
<td>Glucose oxidase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7016</td>
<td>&nbsp;</td>
<tr>
<td>Guanosine-5&#x2019;-triphosphate (GTP)</td>
<td>&nbsp;</td>
<td>Roche Diagnostic</td>
<td>106399</td>
<td>&nbsp;</td>
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<td>Magnesium Chloride (MgCl2)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>63069</td>
<td>&nbsp;</td>
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<td>Paclitaxel (Taxol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1912</td>
<td>&nbsp;</td>
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<td>1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N&#x2032;-bis(2-ethanesulfonic acid) (PIPES)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Potassium hydroxide (KOH)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P-6310</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide (NaOH)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>480878</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin Alexa Fluor 568 conjugate</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S11226</td>
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cargo loading onto kinesin powered molecular shuttles

细胞进化复杂的分子机器，如驱动蛋白马达蛋白和微管丝，积极支持细胞内的货物运输。而动蛋白尾巴域结合各种货物，动蛋白的头域利用的化学能储存在ATP分子沿着微管晶格步骤。长的，僵硬的微管作为长距离的细胞内运输的轨道。 这些电机和长丝，也可以受雇于微型合成环境中，作为分子航天飞机1的组成部分。在经常使用的设计，马达驱动蛋白锚定通过跟踪它们的尾巴表面，载货元素，这些电机推进服务和功能化微管。这些航天飞机可装载货物，利用生物素链亲和素之间的强和选择性结合。 （生物素标记的微管蛋白，链霉亲和素和生物素的货物）关键部件均为市售。 大厦的经典倒活力测定2，分子航天飞机的建设是在这里详细说明。驱动蛋白马达蛋白被吸附到表面覆膜与酪蛋白，生物素标记的微管蛋白聚合微管，坚持动蛋白，随后与罗丹明标记的链霉涂层。 ATP的浓度保持在subsaturating浓度来实现微管滑行速度的最佳装货3。最后，生物素，荧光标记的纳米球是货物补充。纳米微粒的附着微管作为滑翔坚持到表面的微管和纳米球之间的碰撞的结果。 该协议可以很容易地修改，加载，如生物素化DNA，量子点 5或通过 4-6生物素化抗体的抗原种类繁多的各种货物。

Watch this Scientific Journal Video about Cargo Loading onto Kinesin Powered Molecular Shuttles at JoVE.com

Cargo Loading onto Kinesin Powered Molecular Shuttles

滑翔表面坚持kinesin的马达蛋白可以作为一个纳米级的运输系统的功能化微管组成的分子航天飞机。在这里，一个典型的航天飞机系统的组装描述。

货物装载到驱动蛋白供电分子梭

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

Research

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Biology

Ex vivo Mechanical Loading of Tendon

体外机械装载肌腱

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

科研

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Loading Drosophila Nerve Terminals with Calcium Indicators

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Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

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Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

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This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

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Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

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The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Isolation and Purification of Kinesin from Drosophila Embryos

Isolation and Purification of Kinesin from Drosophila Embryos

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

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Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

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Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

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Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...