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股市日水的解决方案。 7.0使用2 M NaOH溶液调节pH值。 </li><li>一个100毫升的1 M MgCl 2的股票在DD水的解决方案。高压灭菌器的解决方案。 </li><li>添加24.2克的管道和日水约800毫升3.1克KOH颗粒，搅拌至溶解。调节pH值至6.9，使用1 M KOH溶液。加入2毫升0.5 M的EGTA原液和1毫升1 M MgCl 2的原液。用dd水1000毫升的体积。 </li><li>分装成50毫升猎鹰管和冻结在-20 ° C以供将来使用。目前正在使用的BRB80管可在4 ° C或在室温下储存。 </li></ul> 2。氯化镁，氯化镁2（100毫米日水） <ul><li>中日水稀释至终浓度达到100毫米。 </li><li>分装到0.5毫升离心管和储存在-20 ° C（10μL体积），以供将来使用</li></ul> 3。 5&#39; -三磷酸鸟苷二钠，GTP（25毫米，用NaOH调整至7日水，pH值） <ul><li>称量和溶解在DD水和2 M NaOH溶液的pH值调整到7。 </li><li>验证通过测量在260 nm紫外吸收的浓度。 （11.7 × 103米-1厘米-1使用的消光系数）。 </li><li>分装（10μL体积）到0.5毫升离心管，在-20 ° C储存供日后使用。 </li></ul> 4。二甲基亚砜，二甲基亚砜<ul><li>分装（10μL体积）到0.5毫升离心管，在-20 ° C储存供日后使用。 </li></ul> 5。紫杉醇（1毫米在DMSO） <ul><li>称量并溶于通风柜下在DMSO终浓度达到1毫米。 </li><li>分装（20μL体积）到0.5毫升离心管，在-20 ° C储存供日后使用。 </li></ul> 6。 D -（+）葡萄糖（2日水的中号） <ul><li>称，日水溶解，终浓度达到了2米</li><li>分装（20μL体积）到0.5毫升离心管，在-20 ° C储存供日后使用。 </li></ul> 7。葡萄糖氧化酶，（2 mg / ml的BRB80） <ul><li>溶解在BRB80实现终浓度为2毫克/毫升。 </li><li>分装（20μL体积）到0.5毫升离心管，在-20 ° C储存供日后使用。 </li></ul> 8。二硫苏糖醇，DTT（1日水的中号） <ul><li>在DD水溶解通风柜下终浓度达到1米的</li><li>分装（20μL体积）到0.5毫升离心管，在-20 ° C储存供日后使用。 </li></ul> 9。过氧化氢酶（0.8 mg / ml的BRB80） <ul><li>在BRB80溶解在至少2个阶段实现终浓度为0.8毫克/毫升。确定在每个阶段的浓度，并通过测量在276纳米和406纳米（使用3.1 × 10 5米-1厘米-1在276纳米和2.2 × 10 5米-1厘米-1在406 nm处的消光系数的紫外吸收该方程为A = CL）。 </li><li>分装（20μL体积）到0.5毫升离心管，在-20 ° C储存供日后使用。 </li></ul> 10。 5&#39; -三磷酸腺苷，ATP（100毫米MgCl 2的 100MM） <ul><li>准备在DD水原液的100毫米氯化镁2。称取干粉，在这股溶液溶解至终浓度达到100毫米。 </li><li>验证通过测量在260 nm紫外吸收的浓度。 （15.4 x 10 3米-1厘米-1使用的消光系数） 。 </li><li>分装（20μL体积）到0.5毫升离心管，在-20 ° C储存供日后使用。 </li></ul> 11。酪蛋白溶液（20 mg / mL的酪蛋白在BRB80） <ul><li>约3克酪蛋白添加到30毫升50毫升猎鹰管DD水。约1小时的旋涡发展，直到厚厚的一致性解决方案。 </li><li>在约15000克离心30分钟。通过0.5微米和0.2微米针头式过滤器的过滤上清。 </li><li>确定上清的浓度，通过测量紫外吸收在280 nm（使用0.67 毫升毫克-1厘米-1的消光系数）。稀释到20毫克/毫升在BRB80。 </li><li>分装（20微升量）0.5毫升离心管，在-20 ° C储存供日后使用。 </li></ul> 2）标准溶液这些准备一天的实验，实验结束后，应将其丢弃。准备1毫升每个。 1。 BRB80CS0.5 <ul><li>稀BRB80酪蛋白溶液中的终浓度为0.5 mg / mL和存储在冰。这个解决方案是引入流通池之前动蛋白和表面吸附后保留动蛋白的活动。 </li></ul> 2。 BRB80CA <ul><li>准备在0.2 mg / mL的酪蛋白和1毫米ATP BRB80和存放在冰。驱动蛋白是进一步稀释使用这个解决方案引入到流通池之前。 </li></ul> 3。 BRB80T <ul><li>稀紫杉醇BRB80的终浓度为10微米，在室温下存储解决方案。这个解决方案是用于稳定微管。 </li></ul> 4。 BRB80CT <ul><li>准备10μM紫杉醇和0.2 mg / mL的酪蛋白在BRB80并在室温下保存。这是使用抗淬灭和微管的解决方案，以进一步准备。 </li></ul> 5。 BRB80AF <ul><li>准备20毫米D -葡萄糖，20μg/ mL的葡萄糖氧化酶，过氧化氢酶8微克/毫升，10 mM的数码地面电视，和20μMATP在BRB80CT并在室温下保存。这个解决方案是使用稀释的链霉亲和微球和“洗出”流细胞多余的链霉。 kinesin的速度可以控制，通过调整在此解决方案中的ATP浓度。 </li></ul> 3）驱动蛋白的制备<ul><li>表达一种动蛋白组成的野生型，全长果蝇动蛋白重链和C -末端在大肠杆菌中的His - tag的构造和净化使用Ni - NTA柱6。 </li><li>等份（10μL）0.5毫升离心管中，并储存于-80 ° C供日后使用。活跃在这些等分动蛋白浓度约200海里。 </li><li>对于一个典型的实验，淡化kinesin的解决方案，在BRB80CA 20倍。标签KIN20在冰上和存储解决方案。 </li></ul> 4）微管的制备<ul><li>在0.5 ml离心管中，准备25μL生长溶液：4毫米BRB80缓冲区MgCl 2的，1毫米GTP和5％DMSO（V / V） 。 </li><li>添加6.25μL的冻干生物素标记的微管蛋白的20微克等分的解决方案。 </li><li>旋涡，然后放置在热浴在37 ° C时30分钟聚合。稀释100倍轻轻BRB80T和旋涡。标签的解决方案，MT100在室温和存储。 </li><li>设为BRB80AF的MT100 10倍稀释。标签解决方案MT1000。 </li></ul> 5）。链霉亲和纳米微球的解决方案 AlexaFluor568的标记链霉浓度在BRB80AF 100 nm的准备。标签上STV100和存储在冰上。同样，稀释5000倍微球BRB80AF解决方案。标签在冰上NS5000和存储。 6）流动池建设构建一个流通池使用了两片玻璃双面胶带分开的盖玻片。这种流动的细胞约2厘米长，1厘米宽，高100微米，体积约20μL。解决方案是引入流动，从一个侧面，用吸管从其他邪恶了，用滤纸细胞。 7）倒含量大会玻璃表面先涂允许动蛋白，以保留其功能后，吸附与酪蛋白。动蛋白被吸附后，微管介绍，这是由动蛋白举行。然后涂上荧光链霉微管。冲洗掉多余的链霉后，生物素化的聚苯乙烯荧光微球（直径为40纳米）进行了介绍。表面吸附固定纳米微球的碰撞与移动微管，并获得加载到他们（图1）。 下面列出的解决方案和未来的解决方案出台之前，允许的时间流的顺序。 <ul><li> BRB80CS0.5，5分钟</li><li> KIN20，5分钟</li><li> MT1000，5分钟</li><li> STV100，5分钟</li><li> BRB80AF，3X </li><li> NS5000 </li></ul> 8）显微镜。 安装纳米微球的介绍后，立即在显微镜舞台上流动池。在这个实验中，一个Eclipse TE2000 - U荧光显微镜（尼康，梅尔维尔，纽约州）配备了一个100倍油浸物镜（NA 1.45），一个X举120灯（EXFO，安大略省，加拿大）和一个IXON EMCCD相机（ANDOR，南温莎，CT）的使用。一个FITC过滤立方体（＃48001）和TRITC过滤立方体（＃48002，色度，罗金厄姆，VT）用于图像纳米微球及微管respectively底部表面上的流动细胞。曝光时间是0.2秒，而拍摄时间是2秒。 <img src="/files/ftp_upload/2006/2006fig1.jpg" alt="图1" /> 图1。分子航天飞机的示意图 。

<ol>
	<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...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Adenosine-5&#x2019;-triphosphate (ATP)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1049</td>
<td>&nbsp;</td>
<tr>
<td>Biotin tubulin</td>
<td>&nbsp;</td>
<td>Cytoskeleton Inc.</td>
<td>T333</td>
<td>&nbsp;</td>
<tr>
<td>Casein</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-0376</td>
<td>&nbsp;</td>
<tr>
<td>Catalase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-9322</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7528</td>
<td>&nbsp;</td>
<tr>
<td>Dimethylsulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D-8779</td>
<td>&nbsp;</td>
<tr>
<td>Dithiotreitol (DTT)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0610</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<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>
<tr>
<td>Magnesium Chloride (MgCl2)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>63069</td>
<td>&nbsp;</td>
<tr>
<td>Paclitaxel (Taxol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1912</td>
<td>&nbsp;</td>
<tr>
<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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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 ...

cargo-loading-onto-kinesin-powered-molecular-shuttles

Research

JoVE Journal

Biology

10.4K 次观看.  University of Florida. 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。

视频: Cargo Loading onto Kinesin Powered Molecular Shuttles

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

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

生物学

Loading Drosophila Nerve Terminals with Calcium Indicators

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 the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

载入果蝇与神经末梢钙指标

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 ...

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

TRE重组酶的分子进化

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

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

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


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

从MEFs基质胶2：到基质胶从MEFs分裂的胚胎干细胞

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

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

从MEFs基质胶3：传代的胚胎干细胞从基质胶到基质胶

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

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

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

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

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

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)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

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

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

直接成像诱导ER钙有针对性的酯酶染料装载（TED）

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

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a &quot;Humanized&quot; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

生成一个“人性化”<em&gt;果蝇</em&gt; S2细胞敏感的驱动蛋白-5的药理抑制

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

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

使用荧光显微镜驱动蛋白-1的货物的识别

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