Name: turboid-based proximity labeling for in planta identification of protein-protein interaction networks
Uploaded: 2020-05-17T13:00:00
Description: Watch this Scientific Journal Video about TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks at JoVE.com

这项工作得到了国家转基因科学技术计划（2019ZX080-003至Y.Z.）、中国国家自然科学基金（31872637至Y.Z.）和中央大学基础研究基金（2019TC028至Y.Z.）和NSF-IOS-1354434的资助， NSF-IOS-1339185 和 NIH-GM132582-01 到 S.P.D.K.

注： 方法的概述如图1所示。
1. 植物材料制备
<ol><li>在潮湿的土壤中高密度地生长N.本查米亚纳种子，并在16小时光（约75μmol/m2s）和8小时暗光周期在23~25°C的气候室中保持。</li>
	<li>大约1周后，小心地将每只幼苗转移到4'x 4'盆中，并将幼苗放在同一室。</li>
	<li>保持在室内的植物约4周，直到它们成长为4-8的叶子阶段，为随后的?...

<ol>
	<li>Bergg&#229;rd, T., Linse, S., James, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+detection+and+analysis+of+protein-protein+interactions.">Methods for the detection and analysis of protein-protein interactions.</a> Proteomics. 7 (16), 2833-2842 (2007).</li><li>Kim, D. I., Roux, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filling+the+void:+proximity-based+labeling+of+proteins+in+living+cells.">Filling the void: proximity-based labeling of proteins in living cells.</a> Trends in Cell Biology. 26 (11), 804-817 (2016).</li><li>Li, P., Li, J., Wang, L., Di, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+labeling+of+interacting+proteins:+Application+of+BioID+as+a+discovery+tool.">Proximity labeling of interacting proteins: Application of BioID as a discovery tool.</a> Proteomics. 17 (20), (2017).</li><li>Roux, K. J., Kim, D. I., Raida, M., Burke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+promiscuous+biotin+ligase+fusion+protein+identifies+proximal+and+interacting+proteins+in+mammalian+cells.">A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.</a> Journal of Cell Biology. 196 (6), 801-810 (2012).</li><li>Kim, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+nuclear+pore+complex+architecture+with+proximity-dependent+biotinylation.">Probing nuclear pore complex architecture with proximity-dependent biotinylation.</a> Proceedings of the National Academy of Sciences. 111 (24), 2453-2461 (2014).</li><li>Lin, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+proximal+and+interacting+proteins+in+rice+protoplasts+by+proximity-dependent+biotinylation.">Screening of proximal and interacting proteins in rice protoplasts by proximity-dependent biotinylation.</a> Frontiers in Plant Science. 8 (749), (2017).</li><li>Khan, M., Youn, J. Y., Gingras, A. C., Subramaniam, R., Desveaux, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+planta+proximity+dependent+biotin+identification+(BioID).">In planta proximity dependent biotin identification (BioID).</a> Scientific Reports. 8 (1), 9212 (2018).</li><li>Conlan, B., Stoll, T., Gorman, J. J., Saur, I., Rathjen, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rapid+in+planta+BioID+system+as+a+probe+for+plasma+membrane-associated+immunity+proteins.">Development of a rapid in planta BioID system as a probe for plasma membrane-associated immunity proteins.</a> Frontiers in Plant Science. 9 (1882), (2018).</li><li>Macharia, M. W., Tan, W. Y. Z., Das, P. P., Naqvi, N. I., Wong, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity-dependent+biotinylation+screening+identifies+NbHYPK+as+a+novel+interacting+partner+of+ATG8+in+plants.">Proximity-dependent biotinylation screening identifies NbHYPK as a novel interacting partner of ATG8 in plants.</a> BMC Plant Biology. 19 (1), 326 (2019).</li><li>Branon, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+proximity+labeling+in+living+cells+and+organisms+with+TurboID.">Efficient proximity labeling in living cells and organisms with TurboID.</a> Nature Biotechnology. 36 (9), 880-887 (2018).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TurboID-based+proximity+labeling+reveals+that+UBR7+is+a+regulator+of+N+NLR+immune+receptor-mediated+immunity.">TurboID-based proximity labeling reveals that UBR7 is a regulator of N NLR immune receptor-mediated immunity.</a> Nature Communications. 10 (1), 3252 (2019).</li><li>Arora, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+proximity-dependent+biotinylation+approaches+in+different+plant+model+systems.">Establishment of proximity-dependent biotinylation approaches in different plant model systems.</a> bioRxiv. , (2019).</li><li>Kim, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+TurboID-mediated+proximity+labeling+for+mapping+a+GSK3+kinase+signaling+network+in+Arabidopsis.">Application of TurboID-mediated proximity labeling for mapping a GSK3 kinase signaling network in Arabidopsis.</a> bioRxiv. , (2019).</li><li>Mair, A., Xu, S. L., Branon, T. C., Ting, A. Y., Bergmann, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+labeling+of+protein+complexes+and+cell-type-specific+organellar+proteomes+in+Arabidopsis+enabled+by+TurboID.">Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID.</a> eLife. 8, 47864 (2019).</li><li>Yuan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+throughput+Barley+stripe+mosaic+virus+vector+for+virus+induced+gene+silencing+in+monocots+and+dicots.">A high throughput Barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots.</a> PLoS ONE. 6 (10), 26468 (2011).</li><li>McCormac, A. C., Elliott, M. C., Chen, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+production+of+highly+competent+cells+of+Agrobacterium+for+transformation+via+electroporation.">A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation.</a> Molecular Biotechnology. 9 (2), 155-159 (1998).</li><li>Gingras, A. C., Abe, K. T., Raught, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+know+the+neighborhood:+using+proximity-dependent+biotinylation+to+characterize+protein+complexes+and+map+organelles.">Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles.</a> Current Opinion in Chemical Biology. 48, 44-54 (2019).</li><li>Firat-Karalar, E. N., Rauniyar, N., Yates, J. R., Stearns, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+Interactions+among+centrosome+components+identify+regulators+of+centriole+duplication.">Proximity Interactions among centrosome components identify regulators of centriole duplication.</a> Current Biology. 24 (6), 664-670 (2014).</li><li>Shen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+pH+in+the+Arabidopsis+endomembrane+system.">Organelle pH in the Arabidopsis endomembrane system.</a> Molecular Plant. 6 (5), 1419-1437 (2013).</li><li>Bally, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rise+and+rise+of+Nicotiana+benthamiana:+a+plant+for+all+reasons.">The rise and rise of Nicotiana benthamiana: a plant for all reasons.</a> Annual Review of Phytopathology. 56 (1), 405-426 (2018).</li></ol>

TurboID生物体蛋白连体酶是由基于酵母的基于酵母的定向进化产生的BioID10。它比其他PL酶有许多优点。TurboID 允许 PL 应用于其他模型系统，包括苍蝇和蠕虫，其最佳生长温度约为 25°C10。虽然PL方法在动物系统中得到了广泛的应用，但在植物中的应用是有限的。此处描述的协议为在N.benthamiana建立基于TurboID的PL提供了一个分步程序，该示范植物已广泛应用于?...

PPI 是各种细胞过程的基础。识别PPI的传统方法包括酵母-双杂交（Y2H）筛查和免疫沉淀，加上质谱（IP-MS）1。1然而，两者都面临一些缺点。例如，Y2H 筛查要求提供目标植物或动物物种的 Y2H 库。这些图书馆的建设是劳动密集型和昂贵的。此外，Y2H方法在异质单细胞真核生物酵母中执行，这可能不代表高真核细胞的细胞状态。
相比之下，IP-MS在捕获瞬态或弱PPI方面效率较低，也不适合低丰度或高水性的蛋白质。植物信号通路中涉及的许多重要蛋白质，如受体状激酶（RLKs）或NLR系列免疫受体以低水平表达，并经常与其他蛋白质短暂相互作用。因此，它极大地限制了对这些蛋白质调节机制的理解。
最近，基于工程抗坏血球过氧化物酶（APEX）和突变大肠杆菌生物丁加体酶比拉R118G（称为BioID）的接近标记（PL）方法已经开发和用于PPIs2，3，4的研究。2,3,4PL的原理是，感兴趣的目标蛋白与酶融合，酶催化了生物仿生-AMP（生物-AMP）的形成。这些自由生物AMP由PL酶释放，并扩散到目标蛋白附近，允许在估计半径为10 nm5的初级胺处对近端蛋白进行生物微化。
与传统 Y2H 和 IP-MS 方法相比，此方法具有显著优势，例如捕获瞬态或弱 PPI 的能力。此外，PL 允许在其原生细胞环境中标记目标蛋白的近端蛋白。不同的PL酶在应用于不同的系统时有独特的缺点。例如，虽然APEX提供比BioID更高的标记动力学，并成功地应用于哺乳动物系统，但这种方法对过氧化氢（H2O2）的要求使其不适合植物的PL研究。
相反，基于 BioID 的 PL 避免了使用有毒的 H2O2，但标记速度很慢（需要 18-24 h 才能完成生物小化），从而使暂时性 PPI 的捕获效率降低。此外，BioID 高效PL所需的较高孵育温度（37 °C）给一些生物体（如植物4）带来了外部压力。7,8,因此，据报告，,在植物（即水稻原发体、阿拉比多普西和北本查米亚纳）中有限地部署基于BioID的PL。最近描述的TurboID酶克服了APEX和基于BioID的PL的不足，TurboID表现出高活性，使PL在RT10的10分钟内达到。基于TurboID的PL已成功应用于哺乳动物细胞、苍蝇和蠕虫10。最近，我们和其他研究小组独立优化和扩展了基于TurboID的PL用于研究不同植物系统中的PPI，包括N.本查米亚和阿拉迪多普西植物和番茄毛根11，12，13，14。11,12,13,14比较分析表明，与BioID11、14相比，TurboID14在植物中的PL性能更好。它还通过识别一些与NLR免疫受体11的新相互作用来证明基于TurboID的PL在植物中的鲁棒性，NLR免疫受体11是一种蛋白质，其相互作用伙伴通常很难用传统方法获得。
该协议通过描述N.本查米亚纳植物中NLR免疫受体的N端TIR域的相互作用蛋白的识别，从而说明了植物中基于TurboID的PL。该方法可以扩展到任何感兴趣的蛋白质在N.本查米亚纳。更重要的是，它为调查其他植物物种（如阿拉伯、番茄和其他）的PPI提供了重要参考。

<table>
	<tbody>
		<tr>
			<td>721 Spectrophotometer</td>
			<td>Metash, made in China</td>
			<td>Q/SXFZ6</td>
			<td>For OD600 measurement</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma</td>
			<td>A6141-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma</td>
			<td>B4639-1G</td>
			<td>50 mM Stock</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5702</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11697489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxycholic acid</td>
			<td>Sigma</td>
			<td>D2510-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol (DTT)</td>
			<td>VWR Life Science</td>
			<td>0281-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin C1</td>
			<td>Invitrogen</td>
			<td>65001</td>
			<td>For affinity purification</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plate</td>
			<td>Corning</td>
			<td>Costar 3590</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>A144S-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>For Western blot analysis</td>
		</tr>
		<tr>
			<td>Lithium chloride solution(LiCl), 8M</td>
			<td>Sigma</td>
			<td>L7026-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Low speed refrigerated centrifuge</td>
			<td>Zonkia, made in China</td>
			<td>KDC-2046</td>
			<td>For desalting</td>
		</tr>
		<tr>
			<td>Magnesium Chloride, Hexahydrate (MgCl2&#183;6H2O)</td>
			<td>Sigma</td>
			<td>M9272-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic rack</td>
			<td>Invitrogen</td>
			<td>123.21D</td>
			<td>For bead adsorption</td>
		</tr>
		<tr>
			<td>Multiskan FC Microplate Photometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>N07710</td>
			<td>For OD595 measurement</td>
		</tr>
		<tr>
			<td>NP-40 (IGEPAL CA-630)</td>
			<td>Sigma</td>
			<td>I8896-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-HA</td>
			<td>Roche</td>
			<td>11867423001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotational mixer</td>
			<td>Kylin-Bell Lab Instrument</td>
			<td>WH-986</td>
			<td>For IP</td>
		</tr>
		<tr>
			<td>Shock incubator</td>
			<td>Labotery, made in China</td>
			<td>ZQPZ-228</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>D2510-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate(SDS)</td>
			<td>Sigma</td>
			<td>L4390-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-HRP</td>
			<td>Abcam</td>
			<td>ab7403</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Scientific Industries</td>
			<td>G-560E</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-jacket Incubator</td>
			<td>Blue pard, made in China</td>
			<td>GHP-9080</td>
			<td>For Agrobacterium incubation</td>
		</tr>
		<tr>
			<td>Zeba Spin Desalting Column</td>
			<td>Thermo Fisher Scientific</td>
			<td>89893</td>
			<td>For removal of biotin</td>
		</tr>
	</tbody>
</table>

turboid-based proximity labeling for in planta identification of protein-protein interaction networks

使用工程抗坏血球过氧化物酶（APEX）或大肠杆菌生物丁加体比拉（称为BioID）的接近标记（PL）技术已成功用于识别哺乳动物细胞中的蛋白质-蛋白质相互作用（PPI）。然而，基于APEX的PL对过氧化氢（H2O2）的要求、生物素（16~24小时）的孵育时间较长，以及基于BioID的PL的孵育温度（37°C）较高，严重限制了其在植物中的应用。最近描述的基于 TurboID 的 PL 解决了 BioID 和 APEX 的许多限制。TurboID 允许在室温 （RT） 条件下只需 10 分钟即可快速贴贴蛋白质标签。尽管 TurboID 的效用已在动物模型中得到证明，但我们最近发现，与 BioID 相比，基于 TurboID 的 PL 在植物中性能更好，用于标记接近感兴趣的蛋白质的蛋白质。此处提供了一个分步协议，用于使用核苷酸结合白氨酸富一种重复（NLR）蛋白质系列的N端收费/微素-1受体（TIR）域作为模型来识别蛋白质相互作用伙伴。该方法通过亲和力纯化，描述了生物微化蛋白的载体构造、蛋白质表达结构的农业渗透、生物结合处理、蛋白质提取和脱盐、定量和富集。这里描述的协议可以很容易地适应研究尼科蒂亚纳和其他植物物种感兴趣的其他蛋白质。

代表数据，说明基于所述协议的预期结果，改编自张等人11。图 1总结了在 N. benthamiana中执行基于 TurboID 的 PL 的过程。图2显示了渗透的N.本查米亚纳叶中的蛋白质表达和生物微化。图3显示，渗透叶片中的生物微化蛋白质有效富集，用于后续质谱分析。需要注意的是，使用链球菌-C1偶联磁珠浓缩生物?...

Watch this Scientific Journal Video about TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks at JoVE.com

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

这里描述的是一种接近标记方法，用于识别尼科蒂亚娜本查米亚叶组织中NLR免疫受体的TIR域的相互作用伙伴。还提供了一个详细的协议，用于识别其他感兴趣的蛋白质之间的相互作用使用这种技术在尼科蒂亚纳和其他植物物种。

基于 TurboID 的接近标签，用于蛋白质-蛋白质相互作用网络的植物中识别

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

NLR免疫受体在调节植物防御各种病原体方面起着至关重要的作用。我们描述了一种基于 TurboID 的接近标记方法，用于识别包含 NL 的 Toll/白细胞-1 受体域的相互作用伙伴，例如尼科蒂亚娜本thamiana 植物中的免疫受体。该协议为研究其他植物物种中的蛋白质-蛋白质相互作用提供了重要的参考，可以扩展到尼科蒂亚娜本thamiana感兴趣的任何蛋白质。与传统方法相比，基于 TurboID 的接近标记方法具有明显的优势，因为它可用于捕获体内的瞬态或弱蛋白-蛋白质相互作用。首先在潮湿的土壤中高密度地种植N.benthamiana种子。将它们保持在23至25摄氏度的18小时光线和8小时黑暗照度的气候室中。将植物留在室内约四个星期，直到它们长到四到八的叶阶段，以便随后进行农业渗透。构建 TurboID 融合，并将质粒转化为农业细菌，如文本协议中描述的那样。使用一毫升无针注射器渗透到完全成熟的N.benthamiana叶的阿巴克斯表皮的孵化。在过滤后36小时，将200微摩尔生物素渗透到叶子中，并在收获叶组织前再维持植物3至12小时。要收集叶样本，切开花叶底部的渗透叶，去除叶静脉，并在液氮中闪冻组织。用刺和砂浆研磨叶组织，并在零下80摄氏度的15或50毫升猎鹰管中储存粉末。要提取总蛋白，将约0.35克叶粉转移到两毫升管中。在这个过程中，一定要将组织保持在液氮中的低温。在粉末中加入700微升的RIPA裂解缓冲液。将管子漩涡10分钟，将样品留在冰上30分钟，每4至5分钟将管子反转一次。在4摄氏度下将管子以2万倍g离心10分钟。然后收集上一液。通过脱盐柱运行样品，去除释放生物素。拆下柱底部的密封器，并将其放在 50 毫升管中。然后松开盖子，在1000倍的g和4摄氏度的温度下将柱子离心两分钟。将脱盐柱放在新鲜 50 毫升管中，用 5 毫升 RIPA 裂解缓冲液平衡柱三次。在1000倍的g和4摄氏度下离心两分钟，每次丢弃流经。在平衡柱中的树脂顶部加入1.5毫升蛋白质提取物。当提取物进入树脂时，再加入100微升的RIPEA裂解缓冲液。离心柱两分钟，将脱盐样品留在冰上，直到进一步使用。要量化脱盐蛋白提取物，请使用 Bio-Rad 分光光度计测量 OD595。根据梯度 BSA 溶液的值绘制标准曲线，并计算脱盐蛋白样品的浓度。在室温下，用一毫升RIP裂解缓冲液平衡链球菌C1结合磁珠，一分钟。使用磁架吸收珠子三分钟，轻轻吸气溶液。然后重复洗涤一次。将蛋白质提取物转移到平衡磁珠上，在旋转器上隔夜在四摄氏度下孵育管子，以亲和地纯化蛋白质。第二天，用磁架捕捉珠子，吸上一代。接下来，用1.7毫升的洗涤缓冲液清洗珠子，将珠子加入管子，并在转子上孵育8分钟。如前所述，取出上流液，用 1.7 毫升洗涤缓冲液 2 和洗涤缓冲液 2 重复洗涤。加入1.7毫升50毫升三叶草HCL去除洗涤剂。然后将管子放在磁架上，取出上一液。用一毫升 50 毫升 Tris HCL 重复洗涤，然后将珠子转移到新的 1.5 毫升管中。最后，用50毫摩尔碳酸氢盐缓冲液洗六次珠子，每次洗涤5分钟。使用100微升珠进行免疫布洛特分析，以确认生物素化蛋白的富集，并闪冻结样品的其余部分，储存在零下80摄氏度。西方的斑点被用来分析农业渗透的N.benthamiana叶中的蛋白质表达和生物基化。渗透叶中的生物素化蛋白与链球菌C1结合磁珠有效丰富，用于后续质谱分析。不同大小的蛋白质被捕获，西方对富集蛋白质的印迹分析显示涂片带。尝试此过程时，请记住拆下脱盐柱底部的密封器，并在每次离心前松开盖。该协议很容易适应尼科蒂亚娜本thamiana感兴趣的其他蛋白质，也可用于在其他植物物种中扩展TurboID PL。

Research

JoVE Journal

Immunology and Infection

Particle Agglutination Method for Poliovirus Identification

脊髓灰质炎病毒识别粒子凝集法

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

科研

免疫与感染

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Visualizing Dengue Virus through Alexa Fluor Labeling

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

A Method for Labeling Vasculature in Embryonic Mice

标签胚胎小鼠血管的方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

为检验新型抗病毒药物的鉴定对蓝舌病病毒

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...