Name: purification of biotinylated cell surface proteins from rhipicephalus microplus epithelial gut cells
Uploaded: 2017-07-23T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Watch this Scientific Journal Video about Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells at JoVE.com

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>

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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><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 &amp;micro;M cell strainer</td><td>Thermo Fisher</td><td>87791</td><td></td></tr><tr><td>3,3&amp;prime;,5,5&amp;prime;-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 &amp;micro;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&amp;nbsp;</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&amp;rsquo;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&amp;nbsp;</td><td>&amp;nbsp;Olympus&amp;nbsp;</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 &gt;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&amp;rsquo;s Balanced Salt Solution</td><td>Sigma Life Sciences</td><td>H9394</td><td>HBSS &amp;ndash; 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 &amp;ndash; stored at -20 &lt;b&gt;&amp;deg;&lt;/b&gt;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&amp;nbsp;</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

Universidad de Cartagena UDEC

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

A Method of Targeted Cell Isolation via Glass Surface Functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

通过玻璃表面功能靶细胞提取方法

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

科研

癌症研究

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

生物化学

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

生物工程

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

使用基因组-规模CRISPR/Cas9基因屏幕进行细胞表面受体识别

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

遗传学

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

生物学

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

免疫与感染

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

使用生物素化测定原代星形细胞培养物中蛋白质的细胞表面表达和吞噬率

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

神经科学

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Cell-surface Biotinylation Assay

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively sequestered in the cytoplasm. Once within a cell, these surface proteins can be either destroyed or &#8220;recycled&#8221; back to the membrane. The cell surface biotinylation assay provides researchers with a way to study these phenomena. The technique makes use of a derivative of the small molecule biotin, which can label surface proteins and then be chemically cleaved. However, if the surface protein is endocytosed, the biotin derivative will be protected from cleavage. Thus, by analyzing the uncleaved, endocytosed biotin label, scientists can assess the amounts of internalized surface proteins.In this video, we review the concepts behind the biotinylation assay, delving into the chemical structure of the biotin derivative and the mechanism of its cleavage. This is followed by a generalized protocol of the technique, and finally, a description of how researchers are currently using it to study the dynamics of different cell surface proteins.

细胞表面生物素化测定

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively ...

教育

JoVE Science Education

高级生物学

细胞生物学

Assessing the Internalization of  Target Surface Proteins Using a Biotin Derivative

Source: Tham, D. K. L., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55974">Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation.</a> J. Vis. Exp. (2017).
This video demonstrates a method to assess target protein internalization in the mouse cortical astrocytes using biotinylation, followed by cell lysis, streptavidin-based protein extraction, denaturation, and Western blot analysis to confirm successful internalization.

使用生物素衍生物评估靶表面蛋白的内化

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Determining Internalization ...

生物素化细胞表面蛋白的纯化

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