这项工作得到了ANR-10-IDEX-0001-02、LabEx单元'n'Scale ANR-11-LBX-0038和收敛Q-life ANR-17-CONV-0005研究所的支持。CJ由居里研究所支持，法国国家研究机构（ANR）授予ANR-12-BSV2-0007和ANR-17-CE13-0021， 国家癌症学院（INCA）赠款2014-PL BIO-11-ICR-1，以及雷彻切医学基金会（FRM）赠款DEQ20170336756。MMM由Vaincre老年痴呆症基金会赠款FR-16055p支持，并得到法国阿尔茨海默氏症赠款AAP SM 2019 n+2023的支持。JAS 得到了欧盟"地平线 2020"研究和创新计划的支持，该计划由玛丽·斯考多夫斯卡-库里赠款协议第 675737 号和 FRM 赠款 FDT201904008210 提供。SB 得到了 FRM 赠款 FDT201805005465 的支持。
我们感谢Janke实验室的所有成员，特别是J.苏普龙，以及G.拉基西奇（米卡利斯研究所、农业巴黎技术研究所）和A.高特劳（理工学院）在制定议定书期间的帮助。我们要感谢居里动物设施在老鼠繁殖和护理方面所提供帮助。
抗体12G10由J.弗兰克尔和M.纳尔逊开发，来自由NICCHD赞助、爱荷华大学维护的发展研究杂交瘤银行。

<ol>
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作者没有什么可透露的。

这里描述的方法提供了一个平台，从细胞线和单个鼠标大脑中快速产生高质量、具有组装能力的中量图布林。它是基于黄金标准协议的图布林纯化牛脑用于该领域多年16，17。该方法的一个特别优点是使用 HeLa S3 细胞的悬浮培养物，一旦建立，就会产生大量的细胞，同时几乎不需要动手时间。这使得该协议在任何细胞生物学实验室中都相对容易执行，而?...

微管在许多细胞过程中起着关键作用。它们为细胞提供形状，为染色体分离构建髓和线粒体主轴，并作为细胞内运输的轨道。为了执行这些不同的功能，微管以不同的方式组织自己。该领域的一个耐人寻味的问题是了解分子机制，使结构和进化保存的微管能够适应这种过多的组织和功能。一个潜在的机制是微管的多样化，这是由被称为"图布林代码"1，2，3的概念定义。图布林密码包括两个主要组成部分:将α和β图布林基因产物（图布林异构型）的微分结合到微管中，以及图布林翻译后修饰（PTM）。
自20世纪70年代以来，体外重组实验结合不断发展的光显微镜技术，为有关微管特性的重要发现铺平了道路:动态不稳定4和跑步5，以及它们的其他机制和功能6、7、8、9、10、11、12、13、14、15。到目前为止，几乎所有的体外实验都是基于使用重复的聚合和去聚合周期从脑组织中纯化的图布林16，17。虽然从脑组织中提纯赋予了大量（通常克量）获得高质量图布林的优势，但一个重要的缺点是异质性，因为从脑组织中纯化的图布林是不同图布林异种型的混合物，并富含许多图布林PTM。这种异质性使得无法描述特定的图布林PTM或同位素在微管特性和功能控制中的作用。因此，生产具有受控图布林PTM和同质异型成分的组装能力图布林对于解决图布林代码的分子机制至关重要。
最近，一种利用酵母Stu2p的微管结合托格（肿瘤过度表达基因）域的亲和色谱净化图布林的方法已经开发出来。在这种方法中，细胞或组织粗裂解的图布林通过一个柱子，它与矩阵固定的托格域结合，从而能够分析给定甚至非常小的样品的整个图布林池。近年来，人们还描述了一种期待已久的净化重组管蛋白的方法。它基于巴库洛病毒系统，其中含有α和β-图布林基因的双柠檬载体在昆虫细胞19中表达。然而，这种方法是非常繁琐和耗时的，因此主要用于研究图布林突变的影响20和图布林等型21，22，23体外。
在目前的协议中，我们描述了一种方法，它使用成熟和广泛使用的聚合去聚化方法作为蓝图，从细胞系或小鼠脑组织24产生不同程度的修饰图布林。在此过程中，图布林在可溶性（4 °C的图布林二毛钱）和聚合形式（在瓜诺辛 5'-三磷酸 [GTP] 存在的情况下在 30 °C 处的微管）之间循环。每种形式都通过连续的离心步骤分离:在冷（4 °C）旋转后，图布林调光器将留在超自然体中，而微管将在30°C下喷出。 此外，在高管氨酸-N，N+-bis（2-乙醇离子酸）（PIPES）浓度下进行一个聚合步骤，允许从微管中去除微管相关蛋白质，从而从最终纯化的图布林中去除。从 HeLa S3 细胞中纯化为悬浮或粘附培养物的 Tubulin 几乎没有任何图布林 PTM，并已用于最近的体外重组实验25，26，27，28.我们进一步调整了从单只小鼠大脑中净化图布林的方法，可用于大量小鼠模型，这些模型具有图布林异种类型和PTM的变化。
在协议中，我们首先描述了源材料（细胞质量或脑组织）的生成，其裂解（图1A），然后是图布林聚合和去聚合的连续步骤，以净化图布林（图1B）。我们进一步描述了评估纯化吐布林的纯度（图2A，B）和数量（图3A，B）的过程。该方法可以通过在图布林纯化（图4B）之前在细胞中过度表达修改酶来生产富含选PTM的图布林。或者，在净化过程中，可以将调子蛋白修改酶添加到图布林中。最后，我们可以从缺乏相应图布林修饰酶（图4B）29的小鼠大脑中提炼出缺乏特定同型或PTM的图蛋白。
我们在这里描述的方法有两个主要优点:（一） 它允许在相对较短的时间内生产足够大量的图布林， （ii） 它产生高品质， 纯图布林， 与特定的图布林异型组成或PTM.在本手稿的相关视频中，我们重点介绍了此程序中涉及的一些关键步骤。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#160;M MgCl2&#160;</td>
			<td>Sigma</td>
			<td>#M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>1-L cell culture vessels</td>
			<td>Techne F7610&#160;</td>
			<td></td>
			<td>Used for spinner cultures. Never stir the empty spinner bottles. When spinner bottles are in the cell culture incubator, always keep the lateral valves of spinner bottles slightly open to facilitate the equilibration of media with incubator&#8217;s atmosphere. After use, fill the spinner bottles immediately with tap water to avoid drying of remaining cells on the bottle walls. Wash the bottles with deionised water, add app 200 ml of deionised water and autoclave. Under a sterile cell culture hood remove the water and allow the bottles to dry completely, still under the hood, for several hours. Never use detergents for cleaning the spinner bottles because any trace amounts of the detergent can be deleterious to the cells.</td>
		</tr>
		<tr>
			<td>1.5- and 2-ml tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14-ml round-bottom tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-cm-diameter sterile culture dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml screw-cap tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol&#160;</td>
			<td>Sigma&#160;</td>
			<td>#M3148</td>
			<td>2-mercaptoethanol is toxic and should be used under the hood.</td>
		</tr>
		<tr>
			<td>4-(2-aminoethyl)-benzenesulfonyl fluoride&#160;</td>
			<td>Sigma&#160;</td>
			<td>#A8456</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>5-, 10- 20-ml syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-ml, 10-ml, 25-ml sterile pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml screw-cap tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma</td>
			<td>#A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-tubulin antibody, 12G10&#160;</td>
			<td>Developed by J. Frankel and M. Nelson, obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by the University of Iowa</td>
			<td></td>
			<td>dilution: 1/500</td>
		</tr>
		<tr>
			<td>Anti-glutamylated tubulin antibody, GT335&#160;</td>
			<td>AdipoGen&#160;</td>
			<td>#AG-20B-0020</td>
			<td>dilution: 1/20,000</td>
		</tr>
		<tr>
			<td>Aprotinin&#160;</td>
			<td>Sigma&#160;</td>
			<td>#A1153</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance (0.1 &#8211; 10 g)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman 1-l polypropylene bottles&#160;</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>Beckman Avanti J-26 XP centrifuge</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>Biological stirrer&#160;</td>
			<td>Techne MCS-104L&#160;</td>
			<td></td>
			<td>Installed in the cell culture incubator (for spinner cultures), 25 rpm for Hela S3 and HEK 293 cells</td>
		</tr>
		<tr>
			<td>Bis N,N&#8217;-Methylene-Bis-Acrylamide&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#161-0201</td>
			<td></td>
		</tr>
		<tr>
			<td>Blender IKA Ultra-Turrax&#174;&#160;</td>
			<td></td>
			<td></td>
			<td>For lysing brain tissue, use 5-mm probe, with the machine set at power 6 or 7. Blend the brain tissue 2-3 times for 15 s on ice.</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma&#160;</td>
			<td>#A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue&#160;</td>
			<td>Sigma&#160;</td>
			<td>#1.08122</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxypeptidase A (CPA)</td>
			<td>Sigma</td>
			<td>#C9268</td>
			<td>Concentration: 1.7&#160;U/&#181;l</td>
		</tr>
		<tr>
			<td>Cell culture hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator set at 37&#176;C, 5% CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>&#160;Sigma&#160;</td>
			<td>#D8418</td>
			<td>DMSO can enhance cell and skin permeability of other compounds. Avoid contact and use skin and eye protection.</td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#41965062</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT, DL-Dithiothreitol&#160;</td>
			<td>Sigma&#160;</td>
			<td>#D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Euromedex</td>
			<td>#EU0007-C</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma&#160;</td>
			<td>#E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute&#160;</td>
			<td>Fisher Chemical&#160;</td>
			<td>#E/0650DF/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma&#160;</td>
			<td>#F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>French pressure cell press&#160;</td>
			<td>Thermo electron corporation</td>
			<td>&#160;#FA-078A</td>
			<td>with a #FA-032 cell; for lysing big amounts of cells. Set at medium ratio, and the gauge pressure of 1,000 psi (corresponds to 3,000 psi inside the disruption chamber).</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>&#160;VWR Chemicals&#160;</td>
			<td>#24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>#G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP&#160;</td>
			<td>Sigma&#160;</td>
			<td>#G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block&#160;</td>
			<td>Stuart&#160;</td>
			<td>#SBH130D</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells&#160;</td>
			<td></td>
			<td>ATCC&#174; CCL-2&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela S3 cells&#160;</td>
			<td>ATCC</td>
			<td>ATCC&#174; CCL-2.2&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl )</td>
			<td>VWR</td>
			<td>#20252.290</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope&#160;</td>
			<td></td>
			<td></td>
			<td>With fluorescence if cell transfection is to be verified</td>
		</tr>
		<tr>
			<td>Isopropanol&#160;</td>
			<td>VWR&#160;</td>
			<td>#20842.298</td>
			<td></td>
		</tr>
		<tr>
			<td>jetPEI</td>
			<td>Polyplus&#160;</td>
			<td>#101</td>
			<td></td>
		</tr>
		<tr>
			<td>JLA-8.1000 rotor&#160;</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>KOH&#160;</td>
			<td>Sigma&#160;</td>
			<td>#P1767</td>
			<td>KOH is corrosive and causes burns; use eye and skin protection.</td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#25030123</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory centrifuge for 50-ml tubes</td>
			<td>Sigma</td>
			<td>4-16 K&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin&#160;</td>
			<td>Sigma</td>
			<td>#L2884</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-pipettes p2.5, p10, p20, p100, p200 and p1000 and corresponding tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropestles</td>
			<td>Eppendorf</td>
			<td>#0030 120.973</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse brain tissue&#160;</td>
			<td></td>
			<td></td>
			<td>Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE). Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (authorization no. 04395.03 given by National Authority) in compliance with the international guidelines.</td>
		</tr>
		<tr>
			<td>Needles 18G X 1 &#189;&#8221; (1.2 X 38 mm</td>
			<td>Terumo</td>
			<td>#18G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 20G X 1 &#189;&#8221; (0.9 X 38 mm</td>
			<td>Terumo</td>
			<td>#20G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 21G X 4 &#190;&#8221; (0.8 X 120 mm</td>
			<td>B.Braun</td>
			<td>#466 5643</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#15140130</td>
			<td></td>
		</tr>
		<tr>
			<td>pH-meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma&#160;</td>
			<td>#P7626</td>
			<td>PMSF powder is hazardous. Use skin and eye protection when preparing PMSF solutions.</td>
		</tr>
		<tr>
			<td>PIPES&#160;</td>
			<td>Sigma&#160;</td>
			<td>#P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette-boy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotors</td>
			<td>Beckman 70.1 Ti; TLA-100.3; and TLA 55</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE electrophoresis equipment&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#1658001FC</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS, Sodium dodecyl sulphate&#160;</td>
			<td>VWR&#160;</td>
			<td>#442444H</td>
			<td>For preparing Laemmeli buffer&#160;</td>
		</tr>
		<tr>
			<td>SDS, Sodium dodecyl sulphate&#160;</td>
			<td>Sigma&#160;</td>
			<td>#L5750</td>
			<td>For preparing &#39;TUB&#39; SDS-PAGE gels</td>
		</tr>
		<tr>
			<td>Sonicator&#160;</td>
			<td>Branson</td>
			<td>#101-148-070</td>
			<td>Used for lysing cells grown as adherent cultures. Use 6.5 mm diameter probe, set the sonicator at &#8220;Output control&#8221; 1, &#8220;Duty cycle&#8221; 10% and time depending on the cell type used.</td>
		</tr>
		<tr>
			<td>Tabletop centrifuge for 1.5 ml tubes</td>
			<td>Eppendorf</td>
			<td>5417R&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED, N, N, N&#8242;, N&#8242;-Tetramethylethylenediamine&#160;</td>
			<td>Sigma</td>
			<td>#9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichostatin A (TSA)</td>
			<td>Sigma</td>
			<td>#T8552</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma&#160;</td>
			<td>#T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base (Tris)</td>
			<td>Sigma&#160;</td>
			<td>#T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Life Technologies</td>
			<td>#15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotors&#160;</td>
			<td></td>
			<td>TLA-55, TLA-100.3 and 70.1 Ti rotors</td>
			<td>Set at 4&#176;C or 30&#176;C based on the need of the experiment&#160;</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#357448&#160;</td>
			<td>for using with TLA-55 rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#349622</td>
			<td>for using with TLA-100.3 rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#355631&#160;</td>
			<td>for using with 70.1 Ti rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuges</td>
			<td>Beckman</td>
			<td>Optima L80-XP (or equivalent) and Optima MAX-XP (or equivalent)</td>
			<td>Set at 4&#176;C or 30&#176;C based on the need of the experiment&#160;</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath equipped with floaters or tube holders</td>
			<td></td>
			<td></td>
			<td>Set at 30&#176;C&#160;</td>
		</tr>
	</tbody>
</table>

purification of tubulin with controlled posttranslational modifications and isotypes from limited sources by polymerization-depolymerization cycles

微管细胞学研究的一个重要方面是在体外重组实验中对微管行为的研究。它们允许分析微管的内在特性，如动力学，以及它们与微管相关蛋白质（MAPs）的相互作用。"图布林代码"是一个新兴的概念，指向不同的图布林同位素同位素和各种翻译后修改 （PTM） 作为微管属性和功能的调节器。要探索图布林密码的分子机制，必须使用具有特定同位素和PTM的纯化图布林进行体外重组实验。
迄今为止，这在技术上是具有挑战性的，因为脑图布林，这是广泛应用于体外实验，窝藏许多PTM，并有一个定义的异构型组成。因此，我们开发了这种协议，利用聚合和去聚合周期的经典方法，从不同来源、不同同型成分和受控PTM中纯化图布林。与现有基于亲和力纯化的方法相比，该方法产生纯聚合能力强的图布林，因为在连续的净化步骤中，对聚合或去聚合具有抗性的图布林被丢弃。
我们描述从细胞系中提炼的图布林，无论是在悬浮中生长还是作为粘附培养物生长，以及从单一小鼠大脑中生长。该方法首先描述了在悬架和附定设置（裂解步骤）中细胞质量的生成，然后是通过聚合-去聚合周期进行图布林纯化的连续阶段。我们的方法产生图布林，可用于解决图布林代码对微管的内在特性和微管与相关蛋白质相互作用的影响的实验。

该方法的主要目标是生产出质量高、装配能力强的图布林，其数量足以对纯化成分进行反复的体外实验。从这种管蛋白组装的微管可用于重组测定，基于全内部反射荧光（TIRF）显微镜技术与动态或稳定的微管，在实验测试微管动力学，与MAP或分子电机的相互作用，并强制发电的电机25。它们还可用于微管-MAP共聚检测和固态NMR光谱28。
在整?...

Watch this Scientific Journal Video about Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles at JoVE.co

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

本协议使用聚合和去聚合周期描述从中小型来源（如培养细胞或单个小鼠大脑）提纯的图布林。纯化的图布林富含特定的同位素或具有特定的翻译后修饰，可用于体外重组测定，以研究微管动力学和相互作用。

通过聚合-去聚合周期，通过有限来源的受控后翻译修改和异种型对图布林进行净化

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They ...

purification-tubulin-with-controlled-posttranslational-modifications

Research

JoVE Journal

Biochemistry

5.0K 次观看.  Institut Curie. 本协议使用聚合和去聚合周期描述从中小型来源（如培养细胞或单个小鼠大脑）提纯的图布林。纯化的图布林富含特定的同位素或具有特定的翻译后修饰，可用于体外重组测定，以研究微管动力学和相互作用。

视频: Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;:设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

科研

生物化学

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

一种测量方法RNA<em&gt;ñ</em<sup&gt; 6</sup&gt; -methyladenosine细胞和组织修改

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential Scanning Calorimetry &amp;#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

差示扫描量热 - 用于评估蛋白质抗原的热稳定性和构象的方法

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

通过等密度梯度纯化对U937细胞进行细胞分级分离

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

从猪肾和小鼠肝脏中提取和纯化FAHD1蛋白

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

从哺乳动物细胞中纯化泛素化p53蛋白

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...