Name: localization of sumo-modified proteins using fluorescent sumo-trapping proteins
Uploaded: 2019-04-27T15:00:00
Description: Watch this Scientific Journal Video about Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins at JoVE.com

我们要感谢 Kerscher 实验室的所有成员的支持, 感谢 Nathalie Nguyen 对手稿的批评阅读, 并感谢 Lidia Epp 对测序。这项工作得到了英联邦研究商业化基金 MF16-034-LS 的支持。贝利-赫斯顿研究基金和查尔斯中心颁发的 ry 和 ch 奖奖学金为 w & m 学生提供研究支持。

1. 重组 Kumutag-fl 红细胞捕获蛋白在固定组织培养细胞中的 SUMO 检测
<ol><li>在6英寸 TC 板的22毫米圆形盖上生长组织培养细胞, 直至 70%-80% 融合。在6孔板中执行步骤1.2–1.8。</li>
	<li>用 DPBS (Dulbecco 的磷酸盐缓冲盐水) 短暂清洗细胞</li>
	<li>对于固定, 请制备4% 对甲醛 (PFA) 溶液 (在 DPBS 中稀释) 的新鲜溶液。要修复单元格, 请在 DPBS 中向每口井添加2毫升 4% PFA。在室温下将细胞培养20分钟。注: 使用 PFA 的?...

<ol>
	<li>Kerscher, O., Felberbaum, R., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+proteins+by+ubiquitin+and+ubiquitin-like+proteins.">Modification of proteins by ubiquitin and ubiquitin-like proteins.</a> Cell and Developmental Biology. 22, 159-180 (2006).</li><li>Kerscher, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SUMOylation. , 1-11 (2016).</li><li>Hay, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO:+a+history+of+modification.">SUMO: a history of modification.</a> Molecular Cell. 18 (1), 1-12 (2005).</li><li>Fu, 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=Disruption+of+SUMO-specific+protease+2+induces+mitochondria+mediated+neurodegeneration.">Disruption of SUMO-specific protease 2 induces mitochondria mediated neurodegeneration.</a> PLoS Genetics. 10 (10), e1004579-e1004579 (2014).</li><li>Zhang, H., Kuai, X., Ji, Z., Li, Z., Shi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over-expression+of+small+ubiquitin-related+modifier-1+and+sumoylated+p53+in+colon+cancer.">Over-expression of small ubiquitin-related modifier-1 and sumoylated p53 in colon cancer.</a> Cell biochemistry and biophysics. 67 (3), 1081-1087 (2013).</li><li>Wang, 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=SUMO-specific+protease+1+promotes+prostate+cancer+progression+and+metastasis.">SUMO-specific protease 1 promotes prostate cancer progression and metastasis.</a> Oncogene. 32 (19), 2493-2498 (2013).</li><li>Karami, S., 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=Novel+SUMO-Protease+SENP7S+Regulates+&#946;-catenin+Signaling+and+Mammary+Epithelial+Cell+Transformation.">Novel SUMO-Protease SENP7S Regulates &#946;-catenin Signaling and Mammary Epithelial Cell Transformation.</a> Scientific Reports. 7 (1), 46477 (2017).</li><li>Pelisch, F., 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+SUMO-Dependent+Protein+Network+Regulates+Chromosome+Congression+during+Oocyte+Meiosis.">A SUMO-Dependent Protein Network Regulates Chromosome Congression during Oocyte Meiosis.</a> Molecular Cell. 65 (1), 66-77 (2017).</li><li>Zhang, X. 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=SUMO-2/3+modification+and+binding+regulate+the+association+of+CENP-E+with+kinetochores+and+progression+through+mitosis.">SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis.</a> Molecular Cell. 29 (6), 729-741 (2008).</li><li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+crisis:+Blame+it+on+the+antibodies.">Reproducibility crisis: Blame it on the antibodies.</a> Nature News. 521 (7552), 274-276 (2015).</li><li>Wang, Z., Prelich, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+control+of+a+transcriptional+regulator+by+SUMO-targeted+degradation.">Quality control of a transcriptional regulator by SUMO-targeted degradation.</a> Molecular and Cellular Biology. 29 (7), 1694-1706 (2009).</li><li>Li, S. J., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+protease+required+for+cell-cycle+progression+in+yeast.">A new protease required for cell-cycle progression in yeast.</a> Nature. 398 (6724), 246-251 (1999).</li><li>Peek, 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=SUMO+targeting+of+a+stress-tolerant+Ulp1+SUMO+protease.">SUMO targeting of a stress-tolerant Ulp1 SUMO protease.</a> PLoS ONE. 13 (1), e0191391 (2018).</li><li>Edgar, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+13+Blastomere+Culture+and+Analysis.">Chapter 13 Blastomere Culture and Analysis.</a> Methods in Cell Biology. 48, 303-321 (1995).</li><li>Pelisch, F., 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=Dynamic+SUMO+modification+regulates+mitotic+chromosome+assembly+and+cell+cycle+progression+in+Caenorhabditis+elegans.">Dynamic SUMO modification regulates mitotic chromosome assembly and cell cycle progression in Caenorhabditis elegans.</a> Nature Communications. 5 (1), 769 (2014).</li><li>Dillingham, M. S., 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=Fluorescent+single-stranded+DNA+binding+protein+as+a+probe+for+sensitive,+real-time+assays+of+helicase+activity.">Fluorescent single-stranded DNA binding protein as a probe for sensitive, real-time assays of helicase activity.</a> Biophysical Journal. 95 (7), 3330-3339 (2008).</li><li>Ruckman, 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=2'-Fluoropyrimidine+RNA-based+aptamers+to+the+165-amino+acid+form+of+vascular+endothelial+growth+factor+(VEGF165).+Inhibition+of+receptor+binding+and+VEGF-induced+vascular+permeability+through+interactions+requiring+the+exon+7-encoded+domain.">2'-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain.</a> The Journal of Biological Chemistry. 273 (32), 20556-20567 (1998).</li><li>Hughes, D. 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=Generation+of+specific+inhibitors+of+SUMO-1-+and+SUMO-2/3-mediated+protein-protein+interactions+using+Affimer+(Adhiron)+technology.">Generation of specific inhibitors of SUMO-1- and SUMO-2/3-mediated protein-protein interactions using Affimer (Adhiron) technology.</a> Science Signaling. 10 (505), eaaj2005 (2017).</li><li>Lopata, A., 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=Affimer+proteins+for+F-actin:+novel+affinity+reagents+that+label+F-actin+in+live+and+fixed+cells.">Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.</a> Scientific Reports. 8 (1), 6572 (2018).</li><li>Gilbreth, R. N., 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=Isoform-specific+monobody+inhibitors+of+small+ubiquitin-related+modifiers+engineered+using+structure-guided+library+design.">Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (19), 7751-7756 (2011).</li><li>Berndt, A., Wilkinson, K. A., Heimann, M. J., Bishop, P., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+characterization+of+the+properties+of+SUMO1-specific+monobodies.">In vivo characterization of the properties of SUMO1-specific monobodies.</a> Biochemical Journal. 456 (3), 385-395 (2013).</li></ol>

在这里, 我们介绍了 kmUTAG-fl 的使用, 一种重组蛋白, 在固定的哺乳动物细胞和解剖线虫性腺的功能研究 sumo。Kumutag-fl 是一种抗压的泛 sumo 特异性试剂, 可识别和捕获原生 sumo 结合蛋白和 SUMO 链。由于 SUMO 的三级结构是高度保守的, 因此很有可能使用 kmUTAG-fl 试剂来分析来自附加模型和非模型系统的 SUMO 变种。因此, KmUTAG-fl 可能是传统抗体染色协议的一种有用的替代试剂或继发试剂。
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该方法的目的是利用重组的 sumo 捕获 UTAG (uLp 域标记) 蛋白, 促进对 sumo 共轭蛋白的研究和分析。如下文所述, UTAG 可用于替代其他试剂和方法来纯化、检测和可视化 sumo 修饰的蛋白质。根据生长条件, 细胞可能含有数百或数千种蛋白质, 这些蛋白质通过 SUMO 或 SUMO 链进行改性 (审查见 Kerscher 等人, 2006年1和 Kerscher2016 2)。这代表了一个相当大的困难, 具体的 sumo 修饰蛋白的功能分析, 特别是因为只有一小部分的特定的亚模化目标实际上是改性3。除了它们在基本的细胞过程中的作用, 如转录调控, 蛋白质稳态, 对细胞压力的反应, 和染色质重塑有丝分裂和减数分裂;现在已经足够清楚的是, sumo、sumo 修饰蛋白和 sumo 途径成分也有可能成为癌症和神经退行性疾病等疾病的生物标志物4,5,6 ,7。这突出表明, 需要强大、可靠和易于获得的工具和创新方法, 以便在各种细胞和样品中检测和功能分析 summo 修饰的蛋白质。
在许多系统中, sumo 特异性抗体是检测、分离和功能分析 sumo 修饰蛋白8,9的首选试剂。然而, 一些市售的 sumo 特异性抗体价格昂贵, 数量或可用性有限, 容易表现出疯狂的亲和力和交叉反应性,在某些情况下缺乏重现性10。另一种方法是在转化后的细胞和生物体中表达表位标记的 SUMO, 但将表位标记连接到 SUMO 可能会人为地降低其与蛋白质靶标11的共轭。此外, 在评估未转化的细胞或组织时, 表位也没有用处。
Ulp1 是一种保存的 SUMO 蛋白酶, 来自酿酒酵母, 它同时处理 sumo 前体和 desumoylates sumo 共轭蛋白12。我们开发了 UTAG 试剂的基础上的偶然观察, 在 Ulp1 的 SUMO 处理ulp OMAIN (ud) 中催化半胱氨酸 (c80s) 的突变不仅可以防止 sumo 裂解, 而且还可以捕获具有高含量的 sumo 共轭蛋白亲和力12。为了简单起见, 我们将这个盒式端子的 sumoapm 捕获 Ulp1(C580S) 片段称为 UTAG (UD TAG 的缩写)。UTAG 是一种重组的泛 sumo 捕获蛋白, 它是用于分离和检测 sumo 修饰蛋白的抗 sumo 抗体的一种有用替代品。重要的是, 它特别识别本土折叠, 共轭 SUMO, 而不仅仅是一个或几个表位在 SUMO。为了提高 UTAG 的蛋白质稳定性和 SUMO 结合强度, 我们从耐压酵母马夏努斯 (km) 中产生了 UTAG 的变种。Kumutag 紧密地结合了与纳米摩尔亲和力13的 sumo 共轭.此外, kmUTAG 耐高温 (42°c)、还原剂 (5 mM TCEP-Tris(2-羧基乙基) 盐酸磷化氢)、变性剂 (高达 2 M 尿素)、氧化剂 (0.6% 过氧化氢) 和非离子洗涤剂。这种应激耐受在苛刻的净化条件和长时间的孵育时间是有益的, 确保其稳定性和 sumo 捕获活性。然而, 毫不奇怪, KmUTAG 的 Sumo-bapege 活性与半胱氨酸修饰试剂、离子洗涤剂和完全变性的蛋白质提取物不兼容。KmUTAG 的显著亲和力和特性表明, 这种试剂可能成为研究多个物种中的超酰化蛋白质的标准试剂的一部分。
在这里, 我们提供了一个简单的方法来检测哺乳动物细胞和线虫中的 summo 修饰蛋白使用重组荧光 Mcherry-kumutag 融合蛋白 (Kumtag-fl)。

<table>
	<tbody>
		<tr>
			<td>16% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Well Cell Culture Plates</td>
			<td>Genesee Scientific/Olympus Plastics</td>
			<td>25-105</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-545-003</td>
			<td>Used as a secondary antibody for mouse monoclonal antibody</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Fisher Scientific</td>
			<td>Gibco 14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 488 conjugated AffiniPure Goat Anti-Moue IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-485-146</td>
			<td>Used as a secondary antibody for mouse monoclonal antibody</td>
		</tr>
		<tr>
			<td>Fisherbrand Coverglass for Growth Cover Glasses</td>
			<td>Fisherbrand</td>
			<td>12545101</td>
			<td></td>
		</tr>
		<tr>
			<td>FLUORO-GEL II with DAPI</td>
			<td>Electron Microscopy Sciences</td>
			<td>50-246-93)</td>
			<td>Mounting media in step 1.11</td>
		</tr>
		<tr>
			<td>FLUORO-GEL with DABCO</td>
			<td>Electron Microscopy Sciences</td>
			<td>17985-02</td>
			<td>With DAPI added to 1 &#181;g/mL; mounting media in step 2.2.5</td>
		</tr>
		<tr>
			<td>Glycine-HCl</td>
			<td>Fisher BioReagents</td>
			<td>BP3815</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine-HCl</td>
			<td>ACROS Organics</td>
			<td>6000-43-7</td>
			<td></td>
		</tr>
		<tr>
			<td>KmUTAG-fl</td>
			<td>Kerafast</td>
			<td></td>
			<td>KmUTAG reagents are available on Kerafast.com</td>
		</tr>
		<tr>
			<td>Oneblock Western-CL blocking buffer</td>
			<td>Prometheus</td>
			<td>20-313</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, Phosphate Buffered Saline, 10X Solution</td>
			<td>Fisher BioReagents</td>
			<td>BP3994</td>
			<td></td>
		</tr>
		<tr>
			<td>PNT2 cell line</td>
			<td>Sigma-Aldrich</td>
			<td>95012613</td>
			<td>Normal prostate epithelium immortalized with SV40.</td>
		</tr>
		<tr>
			<td>pSPOT1</td>
			<td>(ChromoTek GmbH)</td>
			<td>ev-1</td>
			<td>https://www.chromotek.com/fileadmin/user_upload/pdfs/Datasheets/pSpot1_v1.pdf</td>
		</tr>
		<tr>
			<td>SUMO 6F2</td>
			<td>DSHB</td>
			<td>SUMO 6F2</td>
			<td>SUMO 6F2 was deposited to the DSHB by Pelisch, F. / Hay, R.T. (DSHB Hybridoma Product SUMO 6F2)</td>
		</tr>
		<tr>
			<td>SUMO protease buffer [10x]</td>
			<td></td>
			<td></td>
			<td>500 mM Tris-HCl, pH 8.0, 2% NP-40, 1.5 M NaCl</td>
		</tr>
		<tr>
			<td>SUMO-2 Antibody 8A2</td>
			<td>DSHB</td>
			<td>SUMO-2 8A2</td>
			<td>SUMO-2 8A2 was deposited to the DSHB by Matunis, M. (DSHB Hybridoma Product SUMO-2 8A2)</td>
		</tr>
		<tr>
			<td>TCEP-HCL</td>
			<td>GoldBio</td>
			<td>51805-45-9</td>
			<td>Used as a reducing agent at a concentration of 5mM</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher BioReagents</td>
			<td>9002-93-1</td>
			<td>Used for permeablization at 0.1% in DPBS/PBS(for worms)</td>
		</tr>
	</tbody>
</table>

localization of sumo-modified proteins using fluorescent sumo-trapping proteins

在这里, 我们提出了一种新的方法来研究蛋白质的超一色化及其亚细胞定位在哺乳动物细胞和线虫卵母细胞。该方法利用重组修饰的 sumo 捕获蛋白片段 kmutag, 来源于抗压性酵母kluyveromyces marxianus的 up1 sumo 蛋白酶。我们调整了 Kumutag 的特性, 以便在不使用抗体的情况下研究各种模型系统中的超酰化。对于 SUMO 的研究, 与基于抗体的方法相比, Kumutag 有几个优点。这种抗压力的 sumo 捕获试剂是重组生产的, 它能识别来自许多物种的原生 sumo 异形, 与市售抗体不同的是, 它显示出对自由、非共轭的 SUMO 的亲和力降低。这里显示的代表性结果包括 SUMO 结合物在哺乳动物组织培养细胞和线虫卵母细胞中的定位。

Kumutag-fl 是一种重组的、标记为染色体的聊天捕获蛋白。为了生产 kmUTAG-fl, 我们克隆了一个代码优化的 Mcherry-kumutag 到 pSPOT1 细菌过度表达质粒 (图 1)。诱导后, kmUTAG-fl 蛋白在 Spog-trap 上纯化, 洗脱, 冷冻至进一步使用。为了确保 Kumutag-fl 的 SUMO-trapping 捕获活性, 我们确认了与 SUMO1 共轭珠子的结合和 sumocat 融合蛋白的沉淀 (数据未显示, 但见前面的工作<sup...

Watch this Scientific Journal Video about Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins at JoVE.com

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

SUMO 是一种必需的、高度保守的、类似于泛素的改性剂。在这个协议中, 我们描述了使用一个抗压力重组的 sumo 捕获蛋白 (kmUTAG) 来可视化本地的、未标记的 SUMMO 共轭及其在各种细胞类型中的定位。

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

该协议的意义是使用一种新型荧光SUMO捕获UTAG蛋白来检测各种真核细胞中的蛋白质修饰剂S SUMO。该技术的主要优点是在各种细胞（包括哺乳动物组织培养和线虫性腺）中无抗体检测 SUMO 的直接协议。SUMO在癌症和神经退行性疾病中扮演着重要角色，这突出了对SUMO改性蛋白质的检测和功能分析的可靠工具需求。由于SUMO是高度保守的，荧光相扑捕获UTAG蛋白可用于标记在许多不同的生物体结合SUMO。此视频演示了它在哺乳动物细胞中的使用。此外，全文介绍了它在线虫卵母细胞中的用法。该技术的视觉演示为染色和相扑检测前的细胞处理提供了关键提示。首先，在六孔 TC 板中生长 22 毫米圆形盖滑上选择的组织培养细胞，直到 70 至 80% 汇合。用一毫升的DPBS短暂清洗细胞。要修复细胞，请在每个井中加入两毫升新鲜4%PFA和DPBS。在室温下孵育细胞20分钟。在一毫升 DPBS 中清洗固定细胞三次，5 分钟，同时使板螺母化。为了渗透细胞，在DPBS中用1%的Triton X-100孵育15分钟。和以前一样，再次清洗细胞。用500微升1摩尔甘氨酸HCL pH 2孵育细胞10秒。然后立即用 500 微升 10X SUMO 蛋白酶缓冲器或 SPB 中和 pH。洗完电池后，从井中取下盖滑，放在湿度室中。仅对于 UTAG-FL 染色，将一微克 UTAG-FL 与 100 微升 1X SPB 混合，在管中含有 5 毫摩尔 TCEP。然后，将混合物移液到盖滑的细胞上，在室温下在湿度室中孵育一小时。要清洗盖滑，每个盖单上的移液器 200 微升 DPBS，并留在位 10 分钟。再重复洗涤两次。从上次洗涤中拆下盖滑，然后用一滴安装介质倒置到预清洁的显微镜滑轨上。在显微镜下观察之前，将样品隔夜存放在20摄氏度的冰柜中。使用 DAPI 和德州红的适当筛选器集可视化单元格。有关如何标记 SUMO 和固定线虫性腺的协议，请参阅全文。虽然标记哺乳动物细胞非常简单，但标记线虫卵母细胞需要事先接受性腺解剖培训。使用固定PMT2细胞孵育的KKUTAG-FL显示出明显的核染色。扩散的核污渍和明显的核污都可见。使用与DAPI共同染色确认了核定位。与KmUTAG-FL的SUMO诱捕活动一致，核定位模式让人联想到S相扑23染色。与抗S SUMO23 8A2抗体共同染色证实了KmUTAG-FL与SUMO23信号的共同定位。这验证了KmUTAG-FL在哺乳动物细胞中检测S SUMO23的功效。从卵母细胞产生成人草本分离的性腺 c.埃利根线虫被贴上抗S相扑抗体的标签。与之前的报告一致，抗SSUMO抗体最初被本地化为晚期美生前细胞的核质。然后，当核包络破裂，染色体向元相板移动时，它重新分配给配对同源的中央环复合体。在平行的制剂中，标有KKUTAG-FL的性腺也揭示了类似的模式。当卵母细胞开始并完成核包络分解过程时，KmUTAG-FL从核质标记转向专注于环复合体。这些结果验证了KmUTAG-FL作为分析c中与虫病和相扑相关的过程的有用工具。埃利根和可能的其他线虫。在执行固定时，使用新鲜的甲状甲醛和较短的固定时间保持 SUMO 原生折叠，以便 KmUTAG 能够识别 SUMO 至关重要。由于相扑的三级结构是高度保守的，因此很可能使用KmUTAG-FL试剂分析来自其他模型和非模型系统中的SUMO变体。目前，我们使用这种新型荧光相扑诱捕UTAG蛋白来研究SUMO在细胞应激中的功能。最后，请注意，使用固定性前甲醛的所有步骤都应在实验室安全罩中执行，并且必须正确处理该试剂。

Research

JoVE Journal

Biochemistry

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

科研

生物化学

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

膜蛋白对洗涤剂敏感相互作用的检测

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

如何定量光活化荧光蛋白在大容量和活细胞中的分数

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

聚丙烯酰胺凝胶中蛋白质的荧光银染色

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

用细胞内快速光化学氧化蛋白质对细胞内蛋白质进行特征化

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

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

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

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 ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

优化了炔基脂肪酸类似物的掺入，用于使用点击化学检测脂肪酰化蛋白

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

使用巴库洛病毒表达载体系统生产重组PRMT蛋白

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...