Name: split green fluorescent protein system to visualize effectors delivered from bacteria during infection
Uploaded: 2018-05-24T14:30:00
Description: Watch this Scientific Journal Video about Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection at JoVE.com

这项研究是通过由科学、信息和通信技术和未来规划部 (NRF-2018R1A2A1A05019892) 资助的韩国国家研究基金会 (NRF) 和植物分子育种中心的赠款来支持的。PMBC) 下一代 Biogreen 21 农村发展管理计划 (PJ013201) 向 EP。我们感谢国家环境管理仪器中心的成像中心, 为拍摄提供了共焦显微镜。

注: 所有步骤均在室温下执行, 除非另有说明。
1. 植物材料的制备 (4 周)
<ol><li>为benthamiana工厂准备<ol>
<li>在每个花盆的土面上播种benthamiana的2种子, 用塑料圆顶覆盖托盘, 并允许种子在25摄氏度、60% 湿度生长室中发芽, 其温度为16/8 小时/暗光周期。</li>
		<li>两周后, 挑选出并丢弃每壶中最小的幼苗, 并继续种植在相同的生长条件下, 在1.1.1 发芽的应用。每两天增加1...

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	<li>Melotto, M., Underwood, W., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+stomata+in+plant+innate+immunity+and+foliar+bacterial+diseases.">Role of stomata in plant innate immunity and foliar bacterial diseases.</a> Annu Rev Phytopathol. 46, 101-122 (2008).</li><li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stomata+function+in+innate+immunity+against+bacterial+invasion.">Plant stomata function in innate immunity against bacterial invasion.</a> Cell. 126 (5), 969-980 (2006).</li><li>Toruno, T. Y., Stergiopoulos, I., Coaker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-Pathogen+Effectors:+Cellular+Probes+Interfering+with+Plant+Defenses+in+Spatial+and+Temporal+Manners.">Plant-Pathogen Effectors: Cellular Probes Interfering with Plant Defenses in Spatial and Temporal Manners.</a> Annu Rev Phytopathol. 54, 419-441 (2016).</li><li>Buttner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behind+the+lines-actions+of+bacterial+type+III+effector+proteins+in+plant+cells.">Behind the lines-actions of bacterial type III effector proteins in plant cells.</a> FEMS Microbiol Rev. 40 (6), 894-937 (2016).</li><li>Dewoody, R. S., Merritt, P. M., Marketon, M. 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这里描述的协议用于监测细菌 T3SS 注射的效应蛋白在感染时对宿主植物细胞的准确定位。以前, 分离 GFP 系统被用来作为一个工具, 研究哺乳动物蛋白的亚细胞定位23,36,沙门氏菌T3E 本地化, 和农杆菌VirE2 交付通过 T4SS 到植物细胞37。为了在植物细胞中应用这个系统, 以前的研究使用了一种转基因玉米植株, 组成性表达 GFP1-10 ?...

植物是一种无梗的有机体, 在整个生命周期中遇到许多入侵的病原体, 包括细菌、真菌、病毒、昆虫和线虫。在 phytopathogens 中, 革兰阴性细菌病原体, 如铜绿假单胞菌和青, 通过进入伤口或自然开口, 如气孔和具1, 感染寄主植物。为了成功地将寄主植物殖民化, 细菌病原体已经进化形成了各种致病因子2。当细菌侵入寄主植物时, 它们会注射一系列的毒力蛋白--被称为效应者--直接进入植物细胞, 以促进其致病性。这些效应抑制或调节植物先天免疫, 并操纵宿主细胞过程, 导致细菌生存3。
致病细菌主要使用 T3SS 将效应蛋白直接传递到宿主细胞中4。T3SS 类似于一个分子注射器, 其针状通道连接从支架蛋白结构横跨内部和外部细菌膜到宿主细胞的注射点5。这种 T3SS-mediated 效应器 (T3E) 分泌机制在植物的各种革兰氏阴性细菌病原体以及人类中都保存良好。一个代表性植物病原体,野火pv。西红柿DC3000 hrcC变种通常有缺陷的 T3SS, 限制了植物的生长, 可能是由于这个突变体无法完全抑制植物免疫 (通过注射效应蛋白)6。在转移到宿主细胞后, 效应靶针对宿主细胞系统中重要的各种宿主蛋白, 包括植物防御反应、基因转录、细胞死亡、蛋白酶体、囊泡贩运和激素通路7,8,9,10. 因此, 跟踪宿主细胞中效应蛋白的细胞定位是了解它们在调节植物免疫力方面的作用的一个很有吸引力的目标。
大多数 T3Es 的定位研究都采用了农杆菌-介导的大荧光蛋白在寄主植物9中的过度表达。然而, 其他物种引入的基因的异种表达方法已被证明是错误本地化的, 或者偶尔不起作用的11,12,13。此外, 一些研究显示, 细菌效应在宿主细胞中进行了适当的靶向定位14,15,16,17。因此, 瞬时表达的效应在植物细胞的细胞质可能不是功能上或数量相同的效应者, 由 T3SS 提供的病原体感染18。此外, 大荧光标记与效应蛋白的融合可能会扰乱适当的效应传递和可视化18,19。因此, 这些检测 T3E 功能的方法可能无法充分反映 T3SS-secreted 效应的本土化。
绿色荧光蛋白 (GFP) 由一个11股的β桶组成, 其中包含一个包括生色20的中心链。金都et报道了一个新的分裂-GFP 系统, 由一个小组成部分 (gfp β链 11;GFP11) 和一个大的互补片段 (GFP β链 1-10;GFP1-10)21。片段不荧光自己, 但荧光后, 他们的自我联想, 当两个片断是在密切接近。对于蛋白质折叠效率的优化, gfp 的鲁棒折叠变体,即, sfGFP 和 sfGFP选择, 随后为分割的 gfp 系统20,21,22而开发。最近, sfGFP1-10 可选-sfYFP1-10选择和 sfCFP1-10选择的单个氨基酸突变变种, 可以用 sfGFP11 片段进行重建, 并分别显示黄色和青色荧光, 生成23.此外, sfCherry 是 mCherry 的导数, 可以以与 sfGFP23相同的方式拆分为 sfCherry1-10 和 sfCherry11 片段。
该系统已经适应了标签和跟踪 T3SS 在 HeLa 细胞感染期间使用的效应器从沙门氏菌24.然而, 它以前未被优化为寄主植物细菌病原体系统。最近, 我们优化了基于改进的 sfGFP1-10 可选的分割 GFP 系统, 以监视从野火发送到植物单元25的 T3Es 的亚细胞定位。为了促进 T3Es 对不同亚细胞间的定位研究, 一组转基因的拟南芥植物在不同的亚细胞间隔内生成 sfGFP1-10 可选25. 此外, 携带各种细胞器靶向的 sfGFP1-10 的质粒选择用于农杆菌介导的瞬态过度表达和 T3SS-based 效应传递的 sfGFP11-tagged 向量也产生。各种转基因的拟南芥线和质粒来表达 T3Es 的种子, 可以从材料表26,27中提到的来源获得。
在下面的协议中, 我们描述了一个优化系统, 以监测由细菌在宿主细胞中使用分裂 sfGFP 系统传递的效应器的动力学。表达 sfGFP1-10选择的植物感染转基因假单胞菌携带重组 sfGFP11 质粒导致 sfGFP11-tagged 效应器从假单胞菌交付宿主细胞。因此, 这些蛋白质被重组, 并植物常常将到特定的效应靶室 (s)。假单胞野火pv。西红柿CUCPB5500 菌株, 其中18个效应被删除, 使用, 因为这种菌株显示低或没有细胞死亡, 在A. 南芥和N. benthamianas28。然而, 这里描述的所有材料和步骤可以被替换或修改, 以适应分裂 sfGFP 系统的调查其他生物问题或优化在给定的实验室条件。

<table>
	<tbody>
		<tr>
			<td>Arabidopsis transgenic lines</td>
			<td></td>
			<td></td>
			<td>Park, E., Lee, H. Y., Woo, J., Choi, D. &#38; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td>
		</tr>
		<tr>
			<td>CYTO-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69831</td>
			<td></td>
		</tr>
		<tr>
			<td>NU-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69832</td>
			<td></td>
		</tr>
		<tr>
			<td>PT-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69833</td>
			<td></td>
		</tr>
		<tr>
			<td>MT-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69834</td>
			<td></td>
		</tr>
		<tr>
			<td>PX-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69835</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69836</td>
			<td></td>
		</tr>
		<tr>
			<td>GO-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69837</td>
			<td></td>
		</tr>
		<tr>
			<td>PM-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69838</td>
			<td></td>
		</tr>
		<tr>
			<td>Organelle-targeted sfGFP1-10OPT plasmid</td>
			<td></td>
			<td></td>
			<td>Park, E., Lee, H. Y., Woo, J., Choi, D. &#38; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td>
		</tr>
		<tr>
			<td>CYTO-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97387</td>
			<td></td>
		</tr>
		<tr>
			<td>NU-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97388</td>
			<td></td>
		</tr>
		<tr>
			<td>PT-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97389</td>
			<td></td>
		</tr>
		<tr>
			<td>MT-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97390</td>
			<td></td>
		</tr>
		<tr>
			<td>PX-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97391</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97392</td>
			<td></td>
		</tr>
		<tr>
			<td>GO-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97393</td>
			<td></td>
		</tr>
		<tr>
			<td>PM-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97394</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfCherry1-10</td>
			<td>Addgene</td>
			<td>97403</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfYFP1-10</td>
			<td>Addgene</td>
			<td>97404</td>
			<td></td>
		</tr>
		<tr>
			<td>CYTO-sfCFP1-10</td>
			<td>Addgene</td>
			<td>97405</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">sfGFP11-tagged Gateway compatible vector for T3SS-based effector delivery system</td>
			<td>Park, E., Lee, H. Y., Woo, J., Choi, D. &#38; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td>
		</tr>
		<tr>
			<td>pBK-GW-1-2</td>
			<td>Addgene</td>
			<td>98250</td>
			<td>pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBK-GW-1-4</td>
			<td>Addgene</td>
			<td>98251</td>
			<td>pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBK-GW-2-2</td>
			<td>Addgene</td>
			<td>98252</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBK-GW-2-4</td>
			<td>Addgene</td>
			<td>98253</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-1-2</td>
			<td>Addgene</td>
			<td>98254</td>
			<td>pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-1-4</td>
			<td>Addgene</td>
			<td>98255</td>
			<td>pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-2-2</td>
			<td>Addgene</td>
			<td>98256</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-2-4</td>
			<td>Addgene</td>
			<td>98257</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>Bacterial strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agrobacterium tumefaciens GV3101</td>
			<td></td>
			<td></td>
			<td>Csaba Koncz and Jeff Schell, The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet. 204,383-396 (1986); Resistant to gentamycin (50 ug/ml) and rifampicin (50 ug/ml)</td>
		</tr>
		<tr>
			<td>Pseudomonas syringae pv. Tomato CUCPB5500</td>
			<td></td>
			<td></td>
			<td>Kvitko, B. H. et al. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors. PLoS Pathog. 5 (4) (2009).; Resistant to rifampicin (100 ug/ml)</td>
		</tr>
		<tr>
			<td>Media components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plant germination media</td>
			<td></td>
			<td></td>
			<td>Add 2.165g/L Murashige &#38; Skoog powder, 10 g/L sucrose to water. Adjust to pH 5.8 and add 2.2 g/L phytagel. Autocalve.</td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog medium including vitamins</td>
			<td>Duchefa Biochemie</td>
			<td>M0222</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Duchefa Biochemie</td>
			<td>S0809</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytagel</td>
			<td>Sigma-Aldrich</td>
			<td>P8169</td>
			<td></td>
		</tr>
		<tr>
			<td>LB media</td>
			<td></td>
			<td></td>
			<td>Add 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl to water. For solid media, add 15 g/L micro agar. Autoclave.&#160; Allow solution to cool to 55 &#176;C, and add antibiotic if needed.</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>BD Bioscience</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Bioscience</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Duchefa Biochemie</td>
			<td>S0520</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro agar</td>
			<td>Duchefa Biochemie</td>
			<td>M1002</td>
			<td></td>
		</tr>
		<tr>
			<td>King&#39;s B media</td>
			<td></td>
			<td></td>
			<td>10 g/L protease peptone #2, 1.5 g/L anhydrous K2HPO4, 15 g/L of agar to water. Autoclave. Cool down to 55 &#176;C and add sterile 15 ml/L glycerol, 5 ml/L MgSO4 to the medium. Add antibiotics if needed.</td>
		</tr>
		<tr>
			<td>Proteose peptone</td>
			<td>BD Bioscience</td>
			<td>212120</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>1551128 USP</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Duchefa Biochemie</td>
			<td>G1345</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Bioscience</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol-Glutamate (MG) liquid media </td>
			<td></td>
			<td></td>
			<td>Add 10 g/L of mannitol, 2 g/L of L-glutamic acid, 0.5 g/L of KH2PO4, 0.2 g/L of NaCl, and 0.2 g/L of MgSO4 to water. Adjust to pH 7</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Duchefa Biochemie</td>
			<td>M0803</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamic acid</td>
			<td>Duchefa Biochemie</td>
			<td>G0707</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>NIST200B</td>
			<td></td>
		</tr>
		<tr>
			<td>Infiltration buffer</td>
			<td></td>
			<td></td>
			<td>10 mM MES (2-(N-morpholino)-ethane sulfonic acid), 10 mM MgCl2, 150 &#181;M acetosyringone. pH 5.6; Prepare a fresh buffer before use.</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Duchefa Biochemie</td>
			<td>M1503</td>
			<td>Prepare 100 mM (pH 5.6) stock in water. Filter sterilize.</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Prepare 100 mM stock in water. Autoclave.</td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma-Aldrich</td>
			<td>D134406</td>
			<td>Prepare 150 mM stock in DMSO.</td>
		</tr>
		<tr>
			<td>Confocal microscope equipments/materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>710 laser scanning confocal system</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axio observer Z1 inverted microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>ThermoFisher</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
	</tbody>
</table>

split green fluorescent protein system to visualize effectors delivered from bacteria during infection

细菌是各种植物病害中最重要的致病因子之一, 它将一组效应蛋白分泌到宿主植物细胞中, 破坏植物的免疫系统。在感染期间细胞质效应通过 III. 型分泌系统 (T3SS) 传递给宿主细胞质。传递到植物细胞后, 效应器 (s) 靶向特定的隔间, 调节宿主细胞的过程以生存和复制病原体。虽然对宿主细胞中效应蛋白的亚型定位进行了研究, 以了解其在致病性中的作用, 利用荧光蛋白, 直接从细菌中注射的效应研究由于 T3SS 和荧光蛋白之间的不相容性而受到了挑战。
在这里, 我们描述了我们最近的方法, 优化分裂 superfolder 绿色荧光蛋白系统 (sfGFP选择), 以可视化的效应传递通过细菌 T3SS 在宿主细胞。通过 T3SS 分泌的 sfGFP 的 sfGFP11 (11th β链) 可以与特定的细胞器 (sfGFP1-10 可选(1-10 β链 sfGFP) 一起组装, 从而导致现场的荧光发射.该协议提供了一个程序, 以可视化重组 sfGFP 荧光信号的效应蛋白从假单胞菌野火在一个特定的细胞器在拟南芥和烟草 benthamiana植物。

GFP 的β桶结构由十一β链组成, 可分为两个片段, 1-10th链 (GFP1-10 可选) 和 11th (GFP11) 链。尽管两个碎片本身都不是荧光的, 但自组装 sfGFP 可以在接近接近度 (图 1A) 存在时发出荧光。在该系统中, sfGFP1-10 "选择"-表示拟南芥或N. benthamiana植物接种了假单胞菌携带一个 sfGFP11 标记的效应器。由铜绿假单?...

Watch this Scientific Journal Video about Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection at JoVE.com

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

分离绿色荧光蛋白系统可视化细菌在感染过程中传递的效应

以荧光蛋白为基础的方法来监测细菌分泌到宿主细胞中的效应, 这是很有挑战性的。这是由于荧光蛋白与 III. 型分泌系统之间的不相容。在这里, 一个优化的分裂 superfolder GFP 系统是用来可视化的效应, 细菌分泌的宿主植物细胞。

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Research

JoVE Journal

Biology

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

引物延伸捕获：从严重退化的DNA来源有针对性的顺序检索

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

科研

生物学

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

缺乏PMS2，ERCC1，Ku86，视野缺损CcOI在结肠癌进展

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

Single-Molecule Imaging of Nuclear Transport

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

细菌提供的RNAi效应：Transkingdom RNA干扰

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

一种新的澄清方法，以可视化胆道变性在肝癌变态海七鳃鳗（ Petromyzon马里努斯）

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...