作者希望感谢澳大利亚昆士兰州农业和渔业部门提供用于本研究的Rhipicephalus microplus蜱，以及Lucas Karbanowicz协助视频拍摄。

1.从R. microplus肠上皮的解剖<ol><li>在实验当天从牛收集半加成蜱。从主机取出后24小时内解剖。 </li><li>在92 mm x 16 mm培养皿的底部附上一条胶带。在胶带上加一滴超级胶水。将蜱，腹侧放在超级胶上，让其干燥2分钟。 </li><li>将100 mL磷酸盐缓冲盐水（PBS）倒入培养皿中，或直到蜱被完全浸没。 </li><li>使用大小11的手术刀，从眼睛的顶部切割到底部的festoons，在刻度的两边。 </li><li>使用无菌镊子，彻底清除盾板和alloscutum，并暴露出内部器官。 </li><li>去除细白色线状器官（气管）和其他膜，以防止污染。 </li><li>使用镊子去除肠道，通过捏住上部区域并从中拉出尸体。去除任何剩余的肠道组织，确保没有其他组织被解剖。 </li><li>在没有氯化钙和硫酸镁和蛋白酶抑制剂混合物（PIC）的情况下将肠子储存在冰冷的汉克平衡盐溶液（HBSS）中。将干冰中的胆固醇冻结并储存在-20°C。 注意:为了帮助肠道解剖和蜱内脏的详情，请参阅D·索尼希恩，19"蜱的生物学"第3.1章。 </li></ol>上皮细胞分离<ol><li>将解剖的肠倒入50mL管内的70μm细胞过滤器上。 </li><li>用PIC用50 mL冰冷的HBSS冲洗肠道组织，直到溶液清澈透明，胆汁呈现白色/透明的外观。 </li><li>用PIC重新悬挂30 mL冰冷HBSS中的胆固醇，轻轻混匀，并在4℃下以500 xg离心10分钟，以使肠内沉淀。取出上清液，重复洗涤3次。 </li><li）为了去除上皮细胞，重新悬浮在10mL细胞培养基Dulbecco&#39;s改良的Eagle培养基（DMEM），2％胎牛血清（FCS），0.5mM乙二胺四乙酸（EDTA），1mM Tris（2 - 羧乙基）膦（TCEP），PIC，并使用辊以6rpm在37℃下缓慢旋转孵育60分钟。 </li><li>通过250μm的细胞过滤器过滤悬浮液，涡流流过并通过70μm的细胞过滤器过滤，收集剩余的流通。 </li><li>将悬浮液在4℃下以500xg离心20分钟以沉淀单个细胞。 </li></ol> 3.使用密度离心梯度分离单个上皮细胞<ol><li>通过AP15预滤纸过滤，制备密度离心梯度（ 例如 Percoll）。在mqH 2 0（v / v）中制备40％和20％Percoll并在4℃下冷却1小时，然后分层梯度。 </li><li>使用蠕动泵以最低速度设定，将3mL 40％密度离心梯度分离成16mL超速离心管，使其在冰上沉降15分钟。泵的速度应导致每分钟流量&lt;1 mL。 </li><li>将管倾斜成45°角，使用蠕动泵将40％层上的20％密度离心梯度层压。泵的速度应导致每分钟流量&lt;1 mL。允许层在冰上沉降15分钟。 </li><li>在20-40％密度离心梯度下，使用蠕动泵以&lt;1 mL / min流速将3 mL含有蜱肠细胞的DMEM培养基。 </li><li>对离心机进行编程，以实现最大加速和最小减速度以600 xg离心10分钟。收集DMEM之间的间期:20％密度离心梯度和20％:40％密度离心梯度以分离上皮细胞。在4°C储存收集的间期进行后续分析。 </li></ol><html> 4.细胞分离评估<ol><li> 血球 <ol><li>用酒精清洁血球计数器滑块。 </li><li>通过轻轻地上下移动细胞来重悬细胞。移取100μL细胞悬液，置于1.5 mL微量离心管中。 </li><li>加入400μL0.4％台盼蓝。轻轻的轻轻混合管子。 </li><li>移取100μL台盼蓝处理的细胞悬浮液，以缓慢填充血细胞计数器的两个腔室。 </li><li>将血细胞计数器置于光学显微镜下，用10X物镜聚焦于血细胞计数器的网格线上。 </li><li>使用手持计数器，计数一组16个正方形内的活体未染色细胞。在同一个广场内，计数蓝色死亡细胞。继续计数，直到计数了四组16个正方形。 </li><li>使用以下公式计算每毫升的总细胞数: 47eq1.jpg"/&gt; </li><li>使用以下公式计算细胞存活率: <img alt="公式2" src="/files/ftp_upload/55747/55747eq2.jpg" /></li></ol></li><li> 细胞分离可视化 <ol><li>在1.5 mL微量离心管中稀释9μLHBSS中的1μL分离细胞。轻轻地将微量离心管轻轻混匀。 </li><li>将5μLHBSS中的分离细胞移至玻片的中间。用4&#39;，6-二脒基-2-苯基吲哚（DAPI）涂三滴安装介质。 </li><li>在室温下孵育载玻片5分钟。小心地将盖子放在准备物上，避免气泡。 </li><li>在荧光显微镜下观察360nm激发下用DAPI染色的细胞并在460nm发射。 </li></ol></li></ol>细胞表面蛋白生物素化<ol><li>生物素生物素使用生物素（A型）缀合物100μL单细胞上皮细胞试剂盒，按照制造商的说明书，以1:1的表面蛋白与缀合物的摩尔比</li><li>对于细胞裂解，向生物素化细胞中加入100μLPBS，1％Triton X-100,10％甘油，100μM氧化型谷胱甘肽和PIC。在冰上孵育1小时，每10分钟轻轻混匀。 </li><li>将生物素化细胞在4℃下以20,000×g离心20分钟以沉淀不溶物质。收集含有细胞质和生物素化膜蛋白的上清液。 </li><li>使用Bradford测定法确定蛋白质浓度。 </li></ol> 6.生物素化表面蛋白的分离<ol><li>将50μL的链霉亲和素磁珠加入1.5 mL微量离心管中。 </li><li>将管放置在磁性支架中，将珠子收集在管的侧面。取出并弃去上清液。 </li><li>向管中加入1000μLTBS，0.1％Tween-20。轻轻混合，用磁力柱收集珠子天。取出并弃去上清液。 </li><li>将40μg生物素化的细胞表面蛋白结合，用洗涤的磁珠稀释至1×PBS中的300μL。在室温下搅拌孵育2小时。 </li><li>用磁力支架收集珠子，取出并弃去上清液。 </li><li>加入300μLTBS，0.1％Tween-20到管中，轻轻搅拌，重新悬浮珠。收集珠子，取出并弃去上清液。重复此洗涤步骤两次。 </li><li>加入100μL0.1M甘氨酸pH2.0给磁珠，并在室温下孵育5分钟。收集珠子并除去含有洗脱的生物素化表面蛋白质的上清液。 </li><li>在4-20％Tris-MOPS SDS-PAGE凝胶上观察分离的表面蛋白质。 </li></ol> 7.生物素化表面蛋白的评估<ol><li> 点斑 <ol><li>切成7厘米×3厘米的硝酸纤维素膜条。 </li><li>应用10μg总滴答t含量（从1.8以上）和10微克生物素化表面蛋白到膜。在室温下干燥15分钟。 </li><li>转移到容器中并浸没在100mL的封闭缓冲液中。在室温下孵育一小时。丢弃阻塞缓冲区。 </li><li>在100μLPBS，0.05％Tween-20中将硝酸纤维素膜洗涤5分钟，同时搅拌。丢弃洗涤缓冲液并重复洗涤三次。 </li><li>在100μLPBS，0.05％Tween-20中的1/5000链霉亲和素 - 辣根过氧化物酶（HRP）中孵育2小时，并在室温下搅拌，并丢弃任何剩余的溶液。 </li><li>在100μLPBS，0.05％Tween-20中搅拌洗涤硝酸纤维素膜5分钟。丢弃洗涤缓冲液并重复洗涤三次。 </li><li>为了检测，将1片4-氯-1-萘酚溶于10mL冰冷的甲醇中。加入4 mL甲醇原液至20 mL TBS。加入10μL新鲜的30％过氧化氢和immedia适用于硝酸纤维素膜。 </li><li>在室温下搅拌孵育直到底物产生不溶性蓝色终产物。这可能需要2-15分钟。 </li><li>丢弃检测溶液，并在100μLmqH 2 O中洗涤三次。 </li></ol></li><li> ELISA测定 <ol><li>用400μL100mM碳酸盐涂层缓冲液和ELISA板的第一排（A＃）涂层2泳道（平底孔）稀释每个样品200ng， </li><li>将100μL100mM碳酸盐涂层缓冲液加入到行（BH）中的所有其它相应的孔中。 </li><li>通过从A排中的每个孔中移取100μL，并将其转移到B列，准备每个样品的连续稀释液。通过移液轻轻混合，避免产生气泡。每行重复稀释，弃去最后100μL，从排H中的每个孔。 </li><li>用石蜡膜覆盖板，并在4℃下孵育过夜。 </li><li>洗平台e，200μLPBS，0.05％Tween-20 /孔三次。 </li><li>向涂覆的孔中加入200μL封闭缓冲液，用Parafilm覆盖，并在4℃下孵育过夜。 </li><li>在封闭缓冲液中稀释1 / 15,000链霉亲和素-HRP，并向板的每个孔中加入100μL。用石蜡膜覆盖，并在4℃下孵育过夜。 </li><li>在200μLPBS，0.05％Tween-20 /孔中洗涤板5次。 </li><li>要检测，每孔加入100μLTMB试剂。经过充分的显色，通常在10-15分钟之间，加入100μL的1M磷酸停止反应。 </li><li>读取λ= 450nm处各孔的吸光度。 </li></ol></li></ol>

<ol>
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作者没有什么可以披露的。

牛蜱侵染构成了养牛业在世界的热带和亚热带地区的一个主要问题，与控制时使用杀螨剂1，4依赖的最常用的方法。 Bm86进行先前蜱肠上皮表面作为对抗R. microplus侵染10保护性抗原，以有限的成功作为疫苗策略内识别由于Bm86进行地理序列变异和定期升压4的要求。 以前的出版物聚焦在上皮隔?...

扇头蜱microplus，牛蜱，是在对养牛业热带和亚热带地区，因为它矢量牛蜱传热（巴贝虫病），微粒孢子虫病和马焦虫病1，2，3，4经济影响方面最显著外寄生物。努力致力于牛蜱控制，减少有害影响，然而常规方法如使用化学杀螨剂具有隐含的缺点，例如在牛奶和肉中存在化学残留物，以及化学抗性蜱的流行率增加5，6，7。因此，研究了替代蜱控制方法的开发，例如使用天然抗性牛，生物防治（生物杀虫剂）和疫苗分级表4，5，6，7，8，9。 在追求能够被用作候选疫苗的蛋白质时，目前的研究集中在滴滴肠上。中肠壁由沉积在薄基底层上的单层上皮细胞构成，基底层的外侧形成肌肉网络。光和电子显微镜观察表明，中肠由三种类型的细胞组成:储备（未分化），分泌和消化。细胞类型的数量根据生理期有很大差异。分泌和消化细胞都从储备细胞18，19，20发起。 cDNA文库的构建检查蜱肠道的组合物导致了抗原性蛋白质，如Bm86进行的鉴定，作为可能的候选疫苗2，3，4。糖蛋白Bm86位于蜱肠细胞的表面，并在接种疫苗的牛中诱导针对牛蜱（ R. microplus ）的保护性免疫应答。被免疫的宿主产生的抗Bm86IgG被蜱摄入，在蜱肠细胞的表面上识别该抗原，随后扰乱了肠道组织的功能和完整性。基于Bm86抗原的疫苗通过减少雌性哺乳动物的数量，重量和繁殖能力，显示了R. microplus和Rhipicephalus annulatus的有效控制，从而导致后续蜱代4发生减少的幼虫侵袭。然而，基于Bm86的疫苗对于所有的蜱细胞阶段都没有效果表现出对R. microplus的一些地理株不能令人满意的功效，因此在肉牛和奶牛行业都很差采用这些疫苗2,4。 从蜱肠分离上皮细胞的能力是一个重要的创新，这将使研究的进展能够确定不同环境条件下的蛋白质膜组成，包括形态学和生理学。本文所述的方法利用螯合剂乙二胺四乙酸（EDTA）和还原剂三（2-羧乙基）膦（TCEP）从其上皮支持组织10释放上皮。通过振荡机械破坏组织，然后在Percoll中进行不连续梯度离心，回收上皮细胞。本文介绍了一种可行和新颖的用于分离蜱肠道外切的技术血细胞。随后在下游应用如FACS和/或LC-MS / MS分析中分析从这些上皮细胞表面分离的生物素化的细胞表面蛋白质。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4% Trypan Blue</td>
			<td>ThermoFisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>3322</td>
			<td></td>
		</tr>
		<tr>
			<td>100mM Carbonate Buffer</td>
			<td></td>
			<td></td>
			<td>3.03 g Na2CO3, 6.0 g NaHCO3 1000 ml distilled water pH 9.6</td>
		</tr>
		<tr>
			<td>16 mL centrifuge tubes with sealing cap</td>
			<td>Thermo Scientific</td>
			<td>3138-0016</td>
			<td>Cool in ice prior to gradient</td>
		</tr>
		<tr>
			<td>250 &#181;M cell strainer</td>
			<td>Thermo Fisher</td>
			<td>87791</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA</td>
			<td>Sigma</td>
			<td>T0440</td>
			<td>Stored at 4C</td>
		</tr>
		<tr>
			<td>30% Hydrogen Peroxide</td>
			<td>Labscene</td>
			<td>BSPA5.500</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-MOPS Gel</td>
			<td>Gen Script</td>
			<td>M42015</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Chloro-1-naphthol tablet</td>
			<td>Sigma-Aldrich</td>
			<td>C6788</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon Tube</td>
			<td>Corning Blue</td>
			<td></td>
			<td>30 x 115mm style. Polyproplyene conical tube.</td>
		</tr>
		<tr>
			<td>70 &#181;M cell strainer</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>AP15 filter paper</td>
			<td>Millipore</td>
			<td>AO1504200</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin (Type A) Conjugation Kit</td>
			<td>Abcam</td>
			<td>Ab102865</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td></td>
		</tr>
		<tr>
			<td>DP Manager&#160;</td>
			<td>Olympus</td>
			<td>2.2.1.195</td>
			<td>Cell imagery software</td>
		</tr>
		<tr>
			<td>Duct Tape</td>
			<td>Home Handyman</td>
			<td></td>
			<td>48mm x 25mm Duct Tape</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td>DMEM - ice cold for protocol</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Amresco</td>
			<td>0105-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>F96 Maxisorp Immuno Plate</td>
			<td>Nunc</td>
			<td>439454</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12003C</td>
			<td>FCS</td>
		</tr>
		<tr>
			<td>Fluorescence microscope&#160;</td>
			<td>&#160;Olympus&#160;</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>F6057-20ML</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td></td>
			<td>#9 Dumont - Switerzland</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Glycerol for molecular biology &#62;99%</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>410225</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand-Held Counter</td>
			<td>Officeworks</td>
			<td>JA0376230</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Sigma Life Sciences</td>
			<td>H9394</td>
			<td>HBSS &#8211; ice cold for protocol</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Optik Lakor</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>L-Glutathione oxidized</td>
			<td>Sigma-Aldrich</td>
			<td>G4376</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Separation Stand</td>
			<td>Novagen</td>
			<td>-</td>
			<td>4-Tube Magnetic Separation Rack</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>179337</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q Water</td>
			<td>Millipore</td>
			<td>ZRXQ003WW</td>
			<td>Integral Water Purification System for Ultrapure Water</td>
		</tr>
		<tr>
			<td>Nitrocellulose Membrane</td>
			<td>Life Sciences</td>
			<td>66485</td>
			<td>30cm x 3M pure nitrocellulose membrane</td>
		</tr>
		<tr>
			<td>PageRuler Prestained protein Ladder</td>
			<td>Thermo-Fisher</td>
			<td>SM0671</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>1.16 g Na2HPO4, 0.1 g KCl, 0.1 g K3PO4, 4.0 g NaCl (500 ml distilled water) pH 7.4</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Sigma-Aldrich</td>
			<td>P1644-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Masterflex</td>
			<td>7518-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>P6560</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Protein-Free T20 PBS Blocking Buffer</td>
			<td>Thermo-Scientific</td>
			<td>37573</td>
			<td>Stored at 4C. Blocking Buffer</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8215-5ML</td>
			<td>PIC &#8211; stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Quick Start Bradford Dye Reagent 1x</td>
			<td>Biorad</td>
			<td>500-0205</td>
			<td>For Bradford Assay</td>
		</tr>
		<tr>
			<td>Quick Start BSA Standards</td>
			<td>Biorad</td>
			<td>500-0207</td>
			<td>BSA standards for Bradford Assay</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Lab. Co</td>
			<td></td>
			<td>Size 11 Scalpel</td>
		</tr>
		<tr>
			<td>SilverQuest TM Staining Kit</td>
			<td>Invitrogen</td>
			<td>LC6070</td>
			<td></td>
		</tr>
		<tr>
			<td>Simply Blue TM Safe Stain&#160;</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall C6+ Ultracentrifuge</td>
			<td>Thermo Scientific</td>
			<td>46910</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin (HRP)</td>
			<td>Abcam</td>
			<td>AB7403</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Magnetic Beads</td>
			<td>New England Biolabs</td>
			<td>S1420S</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Glue - Ultra Fast Mini</td>
			<td>UHU</td>
			<td></td>
			<td>UHU Super Glue 1mg. Ultra Fast mini</td>
		</tr>
		<tr>
			<td>Table-top Centrifuge</td>
			<td>Eppendorf</td>
			<td>22331</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Thermo Fisher</td>
			<td>20490</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Biorad</td>
			<td>161-0407</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P2287-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Ratek</td>
			<td>VM1</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Grant</td>
			<td>GD100</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of biotinylated cell surface proteins from rhipicephalus microplus epithelial gut cells

Rhipicephalus microplus - 牛蜱 - 是对家畜作为几种病原体的载体的经济影响方面最重要的外寄生物。致力于牛蜱控制以减少其有害影响，重点是发现位于蜱肠上皮细胞表面的疫苗候选物质，例如BM86。目前的研究重点是利用cDNA和基因组文库筛选其他候选疫苗。蜱肠细胞的分离是调查蜱肠细胞膜上表面蛋白质组成的重要优势。本文构建了一种用于分离上皮细胞的新颖可行的方法，从半加入的微量元素的蜱肠内含量。该方案利用TCEP和EDTA从上皮支持组织和不连续密度离心分离器中释放上皮细胞以将上皮细胞与其他细胞类型分开。细胞表面蛋白质生物素化并从蜱肠上皮细胞中分离，使用链霉抗生物素蛋白连接的磁珠，允许在FACS或LC-MS / MS分析中的下游应用。

根据图1所示的原理图，从R. microplus的肠组织中分离上皮细胞。 使用该方案制备的蜱肠上皮细胞的代表性荧光显微镜图像显示在图2A中 和2B。 由于细胞分离是在半富集的R. microplus上进行的，细胞在整个样品中表现为单数，球形，光滑的表面...

Watch this Scientific Journal Video about Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells at JoVE.com

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

一种基于梯度改性密度离心方法被利用来上皮细胞从扇头蜱microplus肠组织隔离。生物素化表面结合蛋白，并通过链霉抗生物素蛋白磁珠纯化，用于下游应用。

生物素化细胞表面蛋白的纯化<emRigicephalus microplus</em&gt;上皮细胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

purification-biotinylated-cell-surface-proteins-from-rhipicephalus

Research

JoVE Journal

Biochemistry

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

9.4K 次观看.  The University of Queensland. 一种基于梯度改性密度离心方法被利用来上皮细胞从扇头蜱microplus肠组织隔离。生物素化表面结合蛋白，并通过链霉抗生物素蛋白磁珠纯化，用于下游应用。 

视频: Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

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 ...

科研

生物化学

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

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 ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

PeptiQuick，将膜蛋白一步进生物仿入肽，用于简化蛋白质结合测定

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

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 ...