Name: extraction and purification of fahd1 protein from swine kidney and mouse liver
Uploaded: 2022-02-18T13:30:00
Description: Watch this Scientific Journal Video about Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver at JoVE.com

作者非常感谢Ayse Öztürk和Eva Albertini的技术援助。用于生成肝组织的小鼠在Univ.-Doz的监督下维持。Pidder Jansen-Dürr博士（因斯布鲁克大学生物医学老龄化研究所，Rennweg 10，6020因斯布鲁克，奥地利）。

所有实验均按照机构指南进行。猪肾是从当地超市新鲜获得的。从因斯布鲁克大学生物医学衰老研究所，Rennweg 10，6020奥地利因斯布鲁克大学生物医学衰老研究所保存的C57BL6野生型小鼠中收获肝脏组织，在Univ.-Doz的监督下。Pidder Jansen-Dürr 博士，2013 年获得项目负责人的道德许可（BMWF-66.008/0007-II/3b/2013）。该项目的小鼠的维护和使用由奥地利教育，科学和研究部（BMBWF）颁发的2020-0.242.978号道德许可...

<ol>
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协议中的关键步骤 遵循处理蛋白质的常见准则至关重要，例如在冰上以及在中等pH和盐条件下工作。使用蛋白酶抑制剂对该方法有益，而强烈建议使用蛋白酶体抑制剂。冷冻和解冻样品可能总是导致蛋白质沉淀（至少部分），因此任何解冻的初始蛋白质裂解物等分试样（步骤2）都应连续处理，不得中断。一般建议解冻后的离心和过滤，以除去微沉淀。
从组织?...

真核FAH结构域蛋白1（FAHD1）作为双功能草酰乙酸（OAA）脱羧酶（ODx）1和酰基丙酮酸水解酶（ApH）2。它定位于线粒体2中，属于酶1，2，3，4，5，6的广泛FAH超家族。虽然其ApH活性仅具有次要相关性，但FAHD1的ODx活性参与TCA循环通量1，7，8，9的调节。OAA不仅是三羧酸循环中柠檬酸盐合酶反应所必需的，而且还作为琥珀酸脱氢酶的竞争性抑制剂，作为电子传递系统的一部分和作为弹力代谢物。人脐静脉内皮细胞（HUVEC）中FAHD1基因表达的下调导致细胞增殖速率10的显着降低，线粒体膜电位的显着抑制，与同时切换到糖酵解有关。工作模型是指线粒体功能障碍相关衰老（MiDAS）11样表型8，其中线粒体OAA水平受到FAHD1活性1，8，9的严格调节。
重组蛋白更容易 通过 细菌12 的表达和纯化而不是从组织中获得。然而，在细菌中表达的蛋白质可能由于缺乏翻译后修饰而偏倚，或者可能只是有问题（即，由于质粒丢失，细菌应激反应，扭曲/未形成的二硫键，无或分泌不良，蛋白质聚集，蛋白水解裂解等）。对于某些应用，蛋白质需要从细胞裂解物或组织中获得，以便包括这种修饰和/或排除可能的伪影。从组织中纯化的蛋白质支持高质量抗体的开发，和/或针对选定酶的有效和特异性药理抑制剂，例如FAHD113。
本手稿介绍了从猪肾和小鼠肝脏中提取和纯化FAHD1的一系列方法。所描述的方法需要快速蛋白质液相色谱（FPLC），但使用常见的实验室设备。替代方法可以在其他地方找到14，15，16，17。在全蛋白提取后，所提出的方案涉及一个测试阶段，其中讨论了硫酸铵沉淀和离子交换色谱的子方案（图1）。在定义了这些亚方案之后，通过使用离子交换和FPLC的尺寸排阻色谱法， 通过 顺序策略提取感兴趣的蛋白质。基于这些指南，最终方案可以单独适应其他感兴趣的蛋白质。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63333/63333fig01.jpg"> 图 1：该协议的总体策略。从上到下：蛋白质是从组织中提取的。制备组织匀浆，离心和过滤。对于每对上清液和颗粒衍生样品，需要进行硫酸铵沉淀和离子交换色谱（FPLC）的测试以探针最佳条件。建立这些子方案后，可以通过硫酸铵沉淀，离子交换色谱和重复大小排阻色谱（FPLC）在不同pH和盐浓度下的顺序程序提取蛋白质。所有步骤都需要通过免疫印迹控制。<a href="https://www.jove.com/files/ftp_upload/63333/63333fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m filter units</td>
			<td>MERCK</td>
			<td>SLGP033RS</td>
			<td>Millex-HP, 0.22 &#181;m, PES 33 mm, not steril</td>
		</tr>
		<tr>
			<td>0.45 &#181;m filter units</td>
			<td>MERCK</td>
			<td>SLHP033NS</td>
			<td>Millex-HP, 0.45 &#181;m, PES 33 mm, not steril</td>
		</tr>
		<tr>
			<td>15 mL Falcon tubes</td>
			<td>VWR</td>
			<td>734-0451</td>
			<td>centrifugal tubes</td>
		</tr>
		<tr>
			<td>50 mL Falcon tubes</td>
			<td>VWR</td>
			<td>734-0448</td>
			<td>centrifugal tubes</td>
		</tr>
		<tr>
			<td>96-Well UV Microplate</td>
			<td>Thermo-Fischer</td>
			<td>8404</td>
			<td>UV/VIS transparent flat-bottom 96 well plates</td>
		</tr>
		<tr>
			<td>Acrylamide/Bis Solution (40%, 29:1 ratio)</td>
			<td>BIO-RAD</td>
			<td>#1610147</td>
			<td>40% acrylamide/bis-acrylamide, 29:1 (3.3% crosslinker) solution for casting polyacrylamide gels</td>
		</tr>
		<tr>
			<td>&#196;KTA FPLC system</td>
			<td>GE Healthcare Life Sciences / Cytiva</td>
			<td>-</td>
			<td>using the FPLC system by GE Healthcare; different custom versions exist; this work used the &#34;&#196;KTA pure&#34; system</td>
		</tr>
		<tr>
			<td>Amicon Ultra-15, PLGC Ultracel-PL Membran, 10 kDa</td>
			<td>MERCK</td>
			<td>UFC901024</td>
			<td>centrifigal filters for protein enrichment; 10 kDa molecular mass filter; 15 mL</td>
		</tr>
		<tr>
			<td>Amicon Ultra-4, PLGC Ultracel-PL Membran, 10 kDa</td>
			<td>MERCK</td>
			<td>UFC801024</td>
			<td>centrifigal filters for protein enrichment; 10 kDa molecular mass filter; 4 mL</td>
		</tr>
		<tr>
			<td>Ammonium sulfate powder</td>
			<td>MERCK</td>
			<td>A4418</td>
			<td>ammonium sulphate for molecular biology, &#8805;99.0%</td>
		</tr>
		<tr>
			<td>Ammoniumpersulfat reagent grade, 98%</td>
			<td>MERCK</td>
			<td>215589</td>
			<td>Catalyst for acrylamide gel polymerization.</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant blue R 250</td>
			<td>MERCK</td>
			<td>1125530025</td>
			<td>Coomassie Brilliant blue R 250 (C.I. 42660) for electrophoresis Trademark of Imperial Chemical Industries PLC. CAS 6104-59-2, pH 6.2 (10 g/l, H2O, 25 &#176;C)</td>
		</tr>
		<tr>
			<td>Dialysis tubing cellulose membrane</td>
			<td>MERCK</td>
			<td>D9277</td>
			<td>Cellulose membranes for the exchange of buffers via dialysis.</td>
		</tr>
		<tr>
			<td>Eppendof tubes 1.5 mL</td>
			<td>VWR</td>
			<td>525-1042</td>
			<td>microcentrifugal tubes; autoclaved</td>
		</tr>
		<tr>
			<td>HiLoad 26/600 Superdex 75 pg</td>
			<td>GE Healthcare Life Sciences / Cytiva</td>
			<td>28989334</td>
			<td>HiLoad Superdex 75 pg prepacked columns are for high-resolution size exclusion chromatography of recombinant proteins</td>
		</tr>
		<tr>
			<td>Immun-Blot PVDF Membrane</td>
			<td>BIO-RAD</td>
			<td>#1620177</td>
			<td>PVDF membranes are protein blotting membranes optimized for fluorescent and multiplex fluorescent applications.</td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Electrophoretic Transfer Cell</td>
			<td>BIO-RAD</td>
			<td>#1703930</td>
			<td>Use the Mini Trans-Blot Cell for rapid blotting of Mini-PROTEAN precast and handcast gels.</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels</td>
			<td>BIO-RAD</td>
			<td>#1658004</td>
			<td>4-gel vertical electrophoresis system, includes electrode assembly, companion running module, tank, lid with power cables, mini cell buffer dam.</td>
		</tr>
		<tr>
			<td>Mono Q 10/100 GL</td>
			<td>GE Healthcare Life Sciences / Cytiva</td>
			<td>17516701</td>
			<td>Mono Q columns are strong anion exchange chromatography columns for protein analysis or small scale, high resolution polishing of proteins.</td>
		</tr>
		<tr>
			<td>Mono S 10/100 GL</td>
			<td>GE Healthcare Life Sciences / Cytiva</td>
			<td>17516901</td>
			<td>Mono S columns are strong cation exchange chromatography columns for protein analysis or small scale high resolution polishing of proteins.</td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder, 10 to 180 kDa</td>
			<td>Thermo-Fischer</td>
			<td>26616</td>
			<td>A mixture of 10 blue-, orange-, and green-stained proteins (10 to 180 kDa) for use as size standards in protein electrophoresis (SDS-PAGE) and western blotting.</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo-Fischer</td>
			<td>23225</td>
			<td>A two-component, high-precision, detergent-compatible protein assay for determination of protein concentration.</td>
		</tr>
		<tr>
			<td>Sonifier 250; Ultrasonic Cell Disruptor w/ Converter</td>
			<td>Branson</td>
			<td>-</td>
			<td>New models at https://www.emerson.com/documents/automation/brochure-sonifier-sfx250-sfx550-cell-disruptors-homogenizers-branson-en-us-168180.pdf</td>
		</tr>
		<tr>
			<td>Swine Anti-Rabbit Immunoglobulins/HRP (affinity isolated)</td>
			<td>Agilent Dako</td>
			<td>P0399</td>
			<td>The antibody used for horseradish peroxidase conjugation reacts with rabbit immunoglobulins of all classes.</td>
		</tr>
		<tr>
			<td>TEMED, 1,2-Bis(dimethylamino)ethane, TMEDA</td>
			<td>MERCK</td>
			<td>T9281</td>
			<td>TEMED (N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine) is molecule which allows rapid polymerization of polyacrylamide gels.</td>
		</tr>
		<tr>
			<td>Tube Roller</td>
			<td>-</td>
			<td>-</td>
			<td>A general tube rotator roller; e.g. a new model at https://labstac.com/de/Mixer/Roller/c/71</td>
		</tr>
		<tr>
			<td>Tube Rotator</td>
			<td>-</td>
			<td>-</td>
			<td>A general tube rotator wheel; e.g. a new model at https://labstac.com/de/Tube-Roller/p/MT123</td>
		</tr>
		<tr>
			<td>ULTRA-TURRAX; T 25 digital</td>
			<td>IKA</td>
			<td>0003725000</td>
			<td>New models at https://www.ika.com/de/Produkte-Lab-Eq/Dispergierer-Dipergiergeraet-Homogenisierer-Homogenisator-csp-177/T-25-digital-ULTRA-TURRAX-cpdt-3725000/</td>
		</tr>
	</tbody>
</table>

extraction and purification of fahd1 protein from swine kidney and mouse liver

富马酰乙酰乙酸水解酶结构域含蛋白1（FAHD1）是真核生物中FAH超家族的第一个成员，在线粒体中充当草酰乙酸脱羧酶。本文介绍了一系列从猪肾和小鼠肝脏中提取和纯化FAHD1的方法。涵盖的方法包括使用快速蛋白质液相色谱 （FPLC） 进行离子交换色谱，使用 FPLC 进行制备和分析凝胶过滤，以及蛋白质组学方法。全蛋白提取后，探讨硫酸铵沉淀和离子交换色谱， 采用 离子交换和尺寸排阻色谱法提取FAHD1。这种具有代表性的方法可以适应其他感兴趣的蛋白质（在显着水平上表达）并针对其他组织进行修饰。从组织中纯化的蛋白质可以支持高质量抗体和/或有效和特异性药理抑制剂的开发。

使用所提出的方案从猪肾和小鼠肝脏中提取FAHD1蛋白。对于小鼠组织，在最后的纯化步骤后需要多个器官才能获得几μg。出于这个原因，本文重点介绍从猪肾脏中提取FAHD1，这是一个更典型的实验。从小鼠肝脏中提取FAHD1是为了呈现该方案的困难和可能的陷阱。通常建议使用显示要纯化的蛋白质的高表达水平的器官。人蛋白图谱22 可能有助于估计模型系统中的表达，或者可以使用?...

Watch this Scientific Journal Video about Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver at JoVE.com

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

该协议描述了如何从猪肾和小鼠肝脏中提取富马酰乙酰乙酸水解酶结构域蛋白1（FAHD1）。所列的方法可以适应于其他感兴趣的蛋白质并针对其他组织进行修饰。

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 ...

从细菌中纯化重组蛋白是一种行之有效的方法。但是，使用这些技术时可能会出现一些问题。拥有从组织中提取蛋白质的方法肯定是有吸引力的。这种方法的主要优点是可以以生理形式提取蛋白质，包括可能影响其折叠和催化活性的所有相互作用。这种方法可以适应其他感兴趣的蛋白质，并针对其他组织进行修饰。从组织中纯化的蛋白质可以支持高质量抗体和药理学抑制剂的开发。首先，通过将250毫升PBS与50毫摩尔氟化钠，1毫摩尔苯基甲基磺酰氟，每毫升抑肽素2微克和1毫摩尔活化正钒酸盐混合来制备蛋白质裂解缓冲液。使用0.22微米注射器过滤单元过滤溶液。每克组织用两毫升裂解缓冲液制备试管，并将其放在冰上。为了制备组织样品，在预先清洁的玻璃板上解剖约100毫克的组织，每个玻璃板放置在聚苯乙烯泡沫盒中的冰上。将组织片段转移到准备好的试管中以进行随后的裂解。为了制备饱和硫酸铵溶液，将500毫升双蒸馏水加热至70摄氏度。搅拌时，逐渐加入硫酸铵粉末，直至不再溶解硫酸铵。将这种饱和溶液冷却至室温，并将其储存在四摄氏度过夜。为了使猪肾组织均质化，优选通过超声探头超声处理悬浮液，同时将样品保持在冰上。对于小鼠肝脏组织，使用电动均质机并定期在PBS中洗涤，以防止堵塞，同时将样品保持在冰上。从样品中取出20微升，并在显微镜下检查匀浆组织的细胞是否被适当破坏。否则，重复均质化。将管子以10， 000倍G在4摄氏度下离心30分钟。将上清液收集在新鲜的管中并将其放在冰上。随后使用0.45微米和0.22微米注射器过滤单元过滤上清液。将上清液等分到10毫升批次中，并在零下20摄氏度冷冻以进行短期储存，或在零下80摄氏度下冷冻以延长储存时间。制备50微升样品用于SDS-PAGE或蛋白质印迹分析，方法是将10微升5倍SDS样品缓冲液加入40微升上清液中，然后在95摄氏度下沸腾10分钟。根据制造商的说明准备不连续的12.5%聚丙烯酰胺SDS-PAGE凝胶。为了进行SDS-PAGE分析，随后用蛋白质标记分子量标准品加载凝胶的孔，5纳克人FAHD1重组蛋白作为阳性对照，20微升待分析的样品，以及剩余的孔和20微升制备的SDS-PAGE样品缓冲液。使用SDS电泳缓冲液在125伏电压下电泳SDS-PAGE凝胶。SDS-PAGE完成后，进行蛋白质印迹分析，使用针对FAHD1的可用抗体探测膜。在制备上清液等分试样如前所述之后，要么在解冻后处理，要么在蛋白质提取后直接处理而不冷冻样品。使用0.22微米过滤装置过滤样品，以排除解冻后可能的沉淀物。在冰上制备六个1.5毫升管，然后将250微升样品转移到每个管中。在制备的试管中制备一系列稀释的硫酸铵，并用蛋白质裂解缓冲液将最终体积组成至1， 000微升。将样品在四摄氏度下在管旋转器上孵育过夜。使用台式离心机，在4摄氏度下以10， 000倍G离心30分钟，并小心地将所有上清液转移到单独的管中。将得到的颗粒风干，并将其中的每一个重悬于1， 000微升的双蒸馏水中。对于上一步中每对重悬的沉淀和上清液，将40微升与10微升的五倍SDS样品缓冲液混合，并在95摄氏度下打开盖子沸腾，直到大部分液体蒸发。然后将沉淀重悬于50%二甲基亚砜的混合物中，在双蒸馏水中。通过成对加载从重悬沉淀和上清液中提取的样品，然后进行蛋白质印迹分析来执行SDS-PAGE。但是，将凝胶在80伏特下电泳三小时。用阴离子或阳离子交换柱设置FPLC系统。用五塔体积的20%乙醇在双蒸馏水中洗涤塔，然后用五塔体积的双蒸馏水洗涤塔。通过进样或使用样品泵将样品涂在色谱柱上，并收集流通量。用一列体积的低盐缓冲液洗涤色谱柱。在三柱体积内设置从100%低盐缓冲液到100%高盐缓冲液的线性梯度洗脱。连续收集一毫升馏分。梯度完成后，继续用高盐缓冲液运行，直到在一柱体积范围内的色谱图中不再检测到蛋白质相关峰。应用一毫升25%SDS溶解在0.5摩尔氢氧化钠和双蒸馏水中以清洁塔。用三塔体积的双蒸馏水和三塔体积的20%乙醇在双蒸馏水中连续洗涤塔。收集所有峰分数和流通量的SDS-PAGE样品。将收集的馏分在液氮中冷冻，并将其储存在零下80摄氏度。通过蛋白质印迹探测它们后，解冻并池化含有目标蛋白质的馏分并丢弃其他部分。为了执行纯化步骤，使用超离心过滤单元将蛋白质溶液的体积减少到两毫升。用0.45微米和0.22微米注射器过滤单元按顺序过滤溶液，以去除任何微沉淀。用一列体积的含有一毫摩尔二硫舒醇的电泳缓冲液平衡尺寸排阻色谱柱。将样品装载到色谱柱上并运行色谱单元，直到如前所述洗脱所有蛋白质。准备50微升样品用于前面所述的SDS-PAGE和蛋白质印迹分析。用一塔体积的双蒸馏水和一塔体积的20%乙醇在双蒸馏水中连续洗涤塔。进行蛋白质印迹分析后，合并所有含有FAHD1的馏分。使用超离心过滤装置将蛋白质溶液的体积减少到两毫升。蛋白质印迹筛选显示上清液中存在猪FAHD1，条带为25千道尔顿，而标记的阳性对照为34千道尔顿。将裂解物中的猪FAHD1通过阴离子交换色谱纯化，并通过洗脱除结合蛋白。通过蛋白质印迹分析鉴定含有猪FAHD1的馏分，合并并浓缩。蛋白质样品由SEC纯化并通过蛋白质印迹分析鉴定。猪FAHD1蛋白的产量约为1毫克，纯度为80%。蛋白质印迹分析检测到小鼠FAHD1蛋白与其他蛋白质一起流出。将流通应用于尺寸排阻色谱。将含有FAHD1的馏分合并，浓缩并应用于SDS-PAGE分析。然而，蛋白质的产量不是很高。猪FAHD1的特异性酶活性随着每总蛋白质FAHD1的相对量的增加而增加，其纯度水平随纯度水平而增加。较高浓度的硫酸铵会引起微笑效应，这种效应可以在较低的电压下解决。进一步在裂解物和上清液中以15%的浓度发现蛋白质。然而，在较高浓度下，样品可能会沉淀并且无法检测到。这里介绍的方案要求蛋白质是可解的。需要测试和定义沉淀和pH的最佳条件或色谱的尺寸调整。当从组织中提取蛋白质而不是从细菌中提取蛋白质时，酶功能的研究，蛋白质结构的研究和抗体的开发得到了极大的支持。

Research

JoVE Journal

Biochemistry

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

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

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

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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 ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

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 ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

PCR 突变体、克隆、表达、快速蛋白质纯化协议和色氨酸合成酶野生类型和突变形式的结晶

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

Ganglioside Extraction, Purification and Profiling

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

用蛋白毒性应激器治疗大 肠杆菌 后蛋白质聚合物的提取和可视化

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...