Name: single-molecule analysis of sf9 purified superprocessive kinesin-3 family motors
Uploaded: 2022-07-27T13:00:00
Description: Watch this Scientific Journal Video about Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors at JoVE.com

V.S. 和 P.S. 感谢 Kristen J. Verhey 教授（密歇根大学，密歇根州安娜堡，美国）和 Roop Mallik 教授（印度理工学院孟买分校 （IITB），印度孟买）在整个研究过程中的无条件支持。附言感谢Sivapriya Kirubakaran博士在整个项目中的支持。V.S.承认通过DBT（赠款号：BT / PR15214 / BRB / 10 / 1449 / 2015和BT / RLF / Re-entry/45 / 2015）和DST-SERB（赠款号：ECR / 2016 / 000913）获得的资金。P.K.N感谢ICMR的资助（拨款号5/13/13/2019/NCD-III）。附言感谢DST的资助（拨款号：SR/WOS-A/LS-73/2017）。D.J.S承认IIT Gandhinagar的奖学金。

1. Sf9培养、转染和病毒生成
注意：将Sf9细胞保持在30mL的Sf-900 / SFM培养基中，在100mL无菌一次性锥形烧瓶中，没有任何抗生素/抗真菌剂，温度为28°C。 将悬浮培养物保持在90rpm的轨道振荡器中。无需供应CO 2 和湿度维护。细胞通常每四天传代培养一次，接种0.5 x 10 6个细胞/mL，以在第4天达到2.0 x 106个细胞/mL的密度。
<ol><li>P0 病毒库存?...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+motor+toolbox+for+intracellular+transport.">The molecular motor toolbox for intracellular transport.</a> Cell. 112, 467-480 (2003).</li><li>Miki, H., Okada, Y., Hirokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+kinesin+superfamily:+insights+into+structure+and+function.">Analysis of the kinesin superfamily: insights into structure and function.</a> Trends in Cell Biology. 15 (9), 467-476 (2005).</li><li>Hirokawa, N., Niwa, S., Tanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motors+in+neurons:+transport+mechanisms+and+roles+in+brain+function,+development,+and+disease.">Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease.</a> Neuron. 68 (4), 610-638 (2010).</li><li>Hirokawa, N., Takemura, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motors+and+mechanisms+of+directional+transport+in+neurons.">Molecular motors and mechanisms of directional transport in neurons.</a> Nature Reviews Neuroscience. 6 (3), 201-214 (2005).</li><li>Patel, N. 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Sf9杆状病毒表达系统是高通量蛋白质生产中最通用和最成功的方法之一19，36，37。Sf9细胞的翻译后修饰能力与哺乳动物系统高度相当15。使用该系统的一个相当大的缺点是它速度慢且对污染敏感。最关键的步骤之一是在Sf9细胞中的有效感染和成功表达，这需要正确处理和维护Sf9细胞而没有任何污染。该系统需?...

极其拥挤的细胞环境给分选注定的蛋白质和分子带来了许多挑战。分子马达和细胞骨架轨迹促进了细胞质内分子组织和时空分布的这种高强度工作量。分子马达是水解ATP等能量货币并在运动和力产生过程中利用该能量的酶1。基于氨基酸序列的相似性，驱动蛋白分为14个家族，尽管有这种相似性，但每个电机都对细胞的功能做出了独特的贡献。Kinesin-3 系列电机是最大的电机之一，包括五个亚家族（KIF1、KIF13、KIF14、KIF16 和 KIF28）2，与多种细胞和生理功能相关，包括囊泡转运、信号传导、有丝分裂、核迁移和发育 3，4，5。驱动蛋白-3转运功能的损害与许多神经退行性疾病，发育缺陷和癌症疾病有关6，7，8，9。
最近的研究表明，与传统的驱动蛋白10，11，12，13相比，驱动蛋白-3电机是单体，但经过货物诱导的二聚化，并导致快速和超加工运动。它们的生化和生物物理表征需要大量纯化的活性蛋白质。然而，它们在原核表达系统中的产生导致无活性或聚集的马达，可能是由于不相容的蛋白质合成，折叠和修饰机制14，15，16，17，18。为了规避这些限制并提高产量，我们在这里建立了一个强大的Sf9杆状病毒表达系统来表达和纯化这些马达。
杆状病毒表达系统使用Sf9昆虫细胞系作为高通量真核重组蛋白表达的宿主系统19，20。杆状病毒具有强大的多面体启动子，有助于异源基因表达和可溶性重组蛋白的产生17。由于其成本效益，操作安全且活性蛋白表达量高，它已成为一种强大的工具21。为了表达感兴趣的蛋白质，关键步骤是生成重组杆粒。由于市售的杆粒生成试剂盒价格昂贵，我们将使用更多样品，因此我们开发了一种内部协议，用于将驱动蛋白-3 电机的大尺寸和小型插入到杆粒中。Sf9纯化的驱动蛋白-3马达用于使用全内反射荧光（TIRF）显微镜表征体外单分子和多马达微管滑动特性。电机用 3 串联荧光分子 （3xmCit） 进行 C 端标记，以提供增强的信号并减少光漂白。由于其更高的信噪比，更少的光毒性以及对靠近盖玻片的非常小区域的选择性成像，TIRF成像已被广泛用于体内和体外单分子水平的蛋白质动力学可视化。
本研究讨论了利用Sf9杆状病毒表达系统以及使用TIRF显微镜对马达进行 体外 单分子成像和多马达滑动分析来纯化驱动蛋白-3马达。总之，该研究表明Sf9纯化马达的运动特性与由哺乳动物细胞裂解物制备的马达相同。因此，我们相信Sf9杆状病毒系统可以适应表达和纯化任何感兴趣的运动蛋白。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sf9 culture and transfection materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-FLAG M2 affinity</td>
			<td>Biolegend</td>
			<td>651502</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
			<td>For motility assay and purification</td>
		</tr>
		<tr>
			<td>Cellfectin</td>
			<td>Invitrogen</td>
			<td>10362100</td>
			<td>For Sf9 transfection</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>D5545</td>
			<td>For motility assay mixture</td>
		</tr>
		<tr>
			<td>FLAG peptide</td>
			<td>Sigma</td>
			<td>F3290</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>Preparing lysing Sf9 cells</td>
		</tr>
		<tr>
			<td>IGEPAL CA 630</td>
			<td>Sigma</td>
			<td>I8896</td>
			<td>Preparing lysing buffer for Sf9 cells</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>For motility assay and purification</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>For preparing lysis buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>For preparing lysis buffer</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>For motility purification</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>Kind gift from Dr. Thomas Pucadyil (Indian Institute of Science Education and Research, Pune, India).</td>
			<td></td>
			<td>For baculovirs expression and purification</td>
		</tr>
		<tr>
			<td>Sf9 culture bottles</td>
			<td>Thermo Scientific</td>
			<td>4115-0125</td>
			<td>For suspension culture</td>
		</tr>
		<tr>
			<td>Sf-900/SFM medium (1X)</td>
			<td>Thermo Scientific</td>
			<td>10902-096 -500ml</td>
			<td>For culturing Sf9 cells</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S1888</td>
			<td>Preparing lysing buffer for Sf9 cells</td>
		</tr>
		<tr>
			<td>Unsupplemented Grace&#8217;s media</td>
			<td>Thermo Scientific</td>
			<td>11595030 -500ml</td>
			<td>For Sf9 transfection</td>
		</tr>
		<tr>
			<td>Mirotubule Polymerization and Single molecule assay materails</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A2647</td>
			<td>For motility and gliding assay</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td>For blocking motility chamber</td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C9322</td>
			<td>For motility and gliding assay</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td>For dissolving Rhodamine</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>3777</td>
			<td>For preparing buffers</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>For motility and gliding assay</td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma</td>
			<td>G2133</td>
			<td>For motility and gliding assay</td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td>For microtubule polymerization</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma</td>
			<td>P1767</td>
			<td>Preparing PIPES buffer pH 6.9</td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma</td>
			<td>P6757</td>
			<td>For motility and gliding assay</td>
		</tr>
		<tr>
			<td>Microtubule gliding assay materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>26G&#160; needle</td>
			<td>Dispovan</td>
			<td></td>
			<td>For shearing microtubules</td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma</td>
			<td>C3400</td>
			<td>For microtubule glidning assay</td>
		</tr>
		<tr>
			<td>GFP nanobodies</td>
			<td>Gift from Dr. Sivaraj Sivaramakrishnan (University of Minnesota, USA)</td>
			<td></td>
			<td>For attaching motors to the coverslip</td>
		</tr>
		<tr>
			<td>Rhodamine</td>
			<td>Thermo Scientific</td>
			<td>46406</td>
			<td>For preparing labelling tubulin</td>
		</tr>
		<tr>
			<td>Microscope and other instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5ml, 1.5 and 2-ml microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For Sf9 culture and purification</td>
		</tr>
		<tr>
			<td>10ml&#160; disposable sterile pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For Sf9 culture and purification</td>
		</tr>
		<tr>
			<td>10ul, 200ul, 1ml micropipette tips</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For Sf9 culture and purification</td>
		</tr>
		<tr>
			<td>15ml concal tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For Sf9 culture and purification</td>
		</tr>
		<tr>
			<td>35mm cell culture dish</td>
			<td>Cole Palmer</td>
			<td>15179-39</td>
			<td>For Sf9 culture</td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorious</td>
			<td></td>
			<td>0.01g-300g</td>
		</tr>
		<tr>
			<td>Benchtop orbial shaking incubator</td>
			<td>REMI</td>
			<td></td>
			<td>For Sf9 suspenculture at 28oC</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>EMCCD Andor iXon Ultra 897</td>
			<td></td>
			<td>For TIRF imaging and acquesition</td>
		</tr>
		<tr>
			<td>Double sided tape</td>
			<td>Scotch</td>
			<td></td>
			<td>For making motility chamber</td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Fisherfinest</td>
			<td>12-548-5A</td>
			<td>size; 22X30</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Blue Star</td>
			<td></td>
			<td>For making motility chamber</td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td>Neuation</td>
			<td></td>
			<td>Dissolving paraffin wax</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon Eclipse Ti- U</td>
			<td></td>
			<td>To check protein expression</td>
		</tr>
		<tr>
			<td>Lasers</td>
			<td>488nm (100mW)</td>
			<td></td>
			<td>For TIRF imaging</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>For sample freezing and storage</td>
		</tr>
		<tr>
			<td>Microcapillary loading tip</td>
			<td>Eppendorf</td>
			<td>EP022491920</td>
			<td>For shearing microtubules</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse Ti2-E with DIC set up</td>
			<td></td>
			<td>For TIRF imaging</td>
		</tr>
		<tr>
			<td>Mini spin</td>
			<td>Genetix, BiotechAsia Pvt.Ltd</td>
			<td></td>
			<td>For quick spin</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>100X TIRF objective with 1.49NA oil immersion</td>
			<td></td>
			<td>For TIRF imaging</td>
		</tr>
		<tr>
			<td>Optima UltraCentrifuge XE</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>For protein purification</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH-meter</td>
			<td>Corning</td>
			<td>Coring 430</td>
			<td>To adjust pH</td>
		</tr>
		<tr>
			<td>Pipette-boy</td>
			<td>VWR</td>
			<td></td>
			<td>For Sf9 culture and purification</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>For protein purification</td>
		</tr>
		<tr>
			<td>Sorvall ST8R centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Protein purification</td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For microtubule polymerization</td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman coulter</td>
			<td></td>
			<td>SW60Ti rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman</td>
			<td></td>
			<td>5 mL, Open-Top Thinwall Ultra-Clear Tube, 13 x 51mm</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Neuation</td>
			<td></td>
			<td>Sample mixing</td>
		</tr>
		<tr>
			<td>Wax</td>
			<td>Sigma</td>
			<td>V001228</td>
			<td>To seal motility chamber</td>
		</tr>
	</tbody>
</table>

single-molecule analysis of sf9 purified superprocessive kinesin-3 family motors

复杂的细胞环境给单分子运动分析带来了挑战。然而，成像技术的进步改善了单分子研究，并在检测和理解荧光标记分子的动态行为方面获得了极大的普及。在这里，我们描述了使用全内反射荧光（TIRF）显微镜对驱动蛋白-3家族马达进行体外 单分子研究的详细方法。Kinesin-3是一个大家族，在细胞和生理功能中起着关键作用，从细胞内货物运输到细胞分裂再到发育。我们之前已经证明，使用在哺乳动物细胞中表达马达制备的细胞裂解物，组成型活性二聚体驱动蛋白-3马达在单分子水平上表现出快速和超加工运动，具有高微管亲和力。我们的实验室使用细胞，生化和生物物理方法研究驱动蛋白-3马达及其调节机制，此类研究需要大规模纯化的蛋白质。使用哺乳动物细胞表达和纯化这些马达将是昂贵且耗时的，而在原核表达系统中的表达会导致显着聚集和无活性的蛋白质。为了克服细菌纯化系统和哺乳动物细胞裂解物带来的局限性，我们建立了一个强大的Sf9杆状病毒表达系统来表达和纯化这些马达。驱动蛋白-3 电机用 3 串联荧光蛋白（3xmCitirine 或 3xmCit）进行 C 末端标记，可提供增强的信号并减少光漂白。Sf9纯化蛋白的 体外 单分子和多马达滑动分析表明，驱动蛋白-3马达具有快速和超强的合成性，类似于我们之前使用哺乳动物细胞裂解物的研究。使用这些测定的其他应用包括电机低聚物条件的详细知识、平行生化研究的特定结合伙伴及其动力学状态。

为了使用Sf9杆状病毒表达大规模表达和纯化活性和功能性重组运动蛋白，该系统需要产生稳定携带编码序列的病毒颗粒以感染Sf9细胞。为了实现这一目标，Sf9细胞用编码KIF1A（1-393LZ）-3xmCit-FLAG的重组杆粒转染。72小时后，显着的细胞群显示出绿色荧光蛋白（mCitrine）的表达，细胞和细胞核扩大（图1 和 图2），表明病毒颗粒（P0）的成功生成。这些病毒颗...

Watch this Scientific Journal Video about Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors at JoVE.com

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

本研究详细介绍了使用Sf9杆状病毒表达系统纯化驱动蛋白-3家族成员KIF1A（1-393LZ）。这些纯化的马达的 体外 单分子和多马达滑动分析表现出与哺乳动物细胞裂解物的马达相当的稳健运动特性。因此，Sf9杆状病毒系统适合表达和纯化感兴趣的运动蛋白。

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

该协议提供了一种使用Sf9杆状病毒系统纯化活性分子马达的简单方法。它还详细介绍了单分子运动和多运动滑动测定，以研究运动蛋白的调节机制。所有传统的载体表达Sf9杆状病毒表达和一步亲和纯化，将引导纯活性运动蛋白研究超处理驱动蛋白在单分子和多运动系统中的机理细节。除了驱动蛋白马达外，该方案还可用于研究其他基于细胞骨架的蛋白质（如薯蓣碱和肌球蛋白）的生化和生物物理特性。该协议详细且易于遵循。一些建议是小心处理Sf9细胞，因为它们容易被污染，并遵循协议中提供的注释。视觉演示非常有效，易于遵循和理解关键步骤，特别是对于那些从未进行过这些测定的人。演示该程序的将是Pushpanjali Soppina和Dipeshwari Shewale，他们是与我和Pradeep Naik一起工作的博士生。首先，将细胞接种在35毫升培养皿中，密度为4.5乘以每毫升10至第五个细胞。24小时后，在管中混合一微克杆粒DNA和100微升未补充的格雷斯培养基。在另一个试管中将6微升转染试剂与100微升未补充的Grace培养基混合。小心地将第一管的内容物转移到第二管中。将它们混合并在室温下孵育混合物45分钟。在混合物中加入0.8毫升未补充的格雷斯培养基。从细胞中混合并吸出Sf900 SFM培养基。将准备好的转染培养基滴加在细胞顶部，并将板孵育6小时。然后小心地取出转染混合物。加入两毫升SF900 SFM培养基，并在28摄氏度下进一步孵育48小时。接下来，在倒置荧光显微镜下检查运动蛋白表达。收获带有感染细胞的培养基，并以500G离心五分钟.收集上清液并快速冷冻等分试样。在无菌的100毫升锥形烧瓶中加入10毫升SF900 SFM培养基和1毫升P-零病毒原液。在 28 摄氏度下孵育，以 90 rpm 的速度持续振荡。72小时后，将细胞在15毫升无菌锥形管中以500G旋转5分钟并收集上清液。对于大规模蛋白表达，用1毫升P1原液病毒感染30毫升悬浮培养物。感染后72小时，检查细胞的蛋白质表达，并将细胞收集在无菌的50毫升锥形管中。旋转后，弃去上清液，收集细胞沉淀。要裂解细胞，向细胞沉淀中加入三毫升冰冷的裂解缓冲液。在4摄氏度下以150， 000G旋转细胞裂解物30分钟。收集上清液并将其与40微升50%ANTI-FLAG M2亲和树脂混合。将混合物在 4 摄氏度下孵育三小时，并端到端滚揉。现在，通过在 500 G 下在 4 摄氏度下旋转一分钟来沉淀 FLAG 树脂。用冰冷的洗涤缓冲液洗涤FLAG树脂颗粒三次。第三次洗涤后，小心地排出洗涤缓冲液。接下来，将含有每毫升100微克FLAG肽的70微升洗涤缓冲液添加到树脂沉淀中，并在4摄氏度下孵育过夜，如前所述。旋转后，将含有纯化蛋白质的上清液收集到新鲜管中。按照手稿中的描述制备聚合混合物，并向聚合混合物中加入 10 微管蛋白。将试管转移到加热块中，在 37 摄氏度下预热，并孵育 30 分钟。制备微管稳定缓冲液后，将其加热，然后将其添加到聚合的微管中。一旦运动流通池室准备好，将30微升稀释的微管溶液流过该室。30分钟后，流动40至50微升封闭缓冲液并孵育。准备运动混合物，并向其中加入一微升纯化的马达。充分混合并将溶液流入运动室。密封运动室的两端并在TIRF照明下成像。专注于单个mCitirin标记的电机，以过程方式移动。将罗丹明标记的微管蛋白与未标记的微管蛋白以1比10的比例混合。聚合 30 分钟后，轻轻加入 30 微升预热的微管稳定缓冲液，并在 37 摄氏度下进一步孵育 5 分钟。通过用毛细管加载尖端移液来剪切微管。制备运动流动室后，流动50微升Sf9纯化的GFP纳米抗体并孵育30分钟。通过流动 50 微升块缓冲液来阻塞盖玻片表面。准备50微升的电机混合物。轻轻混合溶液后将其流入腔室并孵育30分钟。用50微升P12-酪蛋白清洗腔室两次。准备滑动测定混合物并轻轻混合，然后将其流入运动室。密封运动室的末端，并在TIRF照明下成像微管滑动。转染48小时后，未转染的细胞看起来很小，圆形且牢固附着。然而，表达蛋白质的转染细胞松散地附着在表面上并显示出增大的细胞直径。72小时后，超过90%的Sf9细胞表达荧光标记的驱动蛋白-3马达KIF1A，细胞松散地结合在表面。从转染的Sf9细胞中纯化驱动蛋白-3运动蛋白。Kymograph描绘了驱动蛋白-3马达沿着微管表面过程性地行走。运动事件显示平均速度和运行长度分别约为每秒2.44微米和10.79微米。Kymograph指示驱动蛋白-3电机平滑的微管滑动，平均速度约为每秒1.37微米。最重要的是在不干扰微管混合物的情况下添加稳定缓冲液，并且不要剪切微管。该协议可用于其他应用，例如ATP酶测量，生物物理分析，机制，体外重建测定等。使用sf9纯化电机，我们最近证明了驱动蛋白-3电机具有快速和超强的加工能力。有趣的是，与表征良好的电机Kinesin-1相比，这些电机表现出较高的ATP周转率。

Research

JoVE Journal

Biochemistry

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

科研

生物化学

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

磁性镊子 quadruplexes 单分子操作

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

用于活细胞超分辨率显微镜和单分子跟踪的传统 BODIPY 结合物

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

单分子荧光能量转移研究核糖体蛋白质合成

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...