Name: in vitro analysis of e3 ubiquitin ligase function
Uploaded: 2021-05-14T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Analysis of E3 Ubiquitin Ligase Function at JoVE.com

我们感谢实验室成员对手稿的批判性讨论和有益的建议。由于大小限制，我们很抱歉没有引用有价值的贡献。这项工作得到了Deutsche Forschungsgemeinschaft（DFG，德国研究基金会）的支持 - SFB 1218 - Projektnumber 269925409和卓越集群EXC 229 / CECAD到TH。这项工作由德国卓越战略（EXC 2030 - 390661388和SFB 1218 - Projektnumber 269925409到T.H. Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft（DFG）资助，用于德国卓越战略 - EXC 2030 - 390661388 und - SFB 1218 - Projektnummer：269925409 an T.H. gefördert。

1. 缓冲液和试剂的制备
注：下面列出了在实验室中手动制备的缓冲液和试剂。实验方案中使用的所有其他缓冲液和试剂均从不同来源购买，并根据制造商的说明使用。
<ol><li>准备10x磷酸盐缓冲盐水（10x PBS）。为此，将1.37 M氯化钠（NaCl），27 mM氯化钾（KCl），80mM磷酸氢二钠二水合物（Na2HPO4 .2H2O）和20 mM磷酸二氢钾（KH2PO4）混合在1升?...

<ol>
	<li>Kuhlbrodt, K., Mouysset, J., Hoppe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchestra+for+assembly+and+fate+of+polyubiquitin+chains.">Orchestra for assembly and fate of polyubiquitin chains.</a> Essays in Biochemistry. 41, 1-14 (2005).</li><li>Pickart, C. M., Eddins, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin:+structures,+functions,+mechanisms.">Ubiquitin: structures, functions, mechanisms.</a> Biochimica et Biophysica Acta. 1695 (1-3), 55-72 (2004).</li><li>Dye, B. T., Schulman, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+mechanisms+underlying+posttranslational+modification+by+ubiquitin-like+proteins.">Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins.</a> Annual Review of Biophysics and Biomolecular Structure. 36, 131-150 (2007).</li><li>Koegl, M., 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+novel+ubiquitination+factor,+E4,+is+involved+in+multiubiquitin+chain+assembly.">A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly.</a> Cell. 96 (5), 635-644 (1999).</li><li>Hoppe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiubiquitylation+by+E4+enzymes:+'one+size'+doesn't+fit+all.">Multiubiquitylation by E4 enzymes: 'one size' doesn't fit all.</a> Trends in Biochemical Sciences. 30 (4), 183-187 (2005).</li><li>Stewart, M. D., Ritterhoff, T., Klevit, R. E., Brzovic, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E2+enzymes:+more+than+just+the+middle+men.">E2 enzymes: more than just the middle men.</a> Cell Research. 26 (4), 423-440 (2016).</li><li>Clague, M. J., Heride, C., Urb&#233;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+demographics+of+the+ubiquitin+system.">The demographics of the ubiquitin system.</a> Trends in Cell Biology. 25 (7), 417-426 (2015).</li><li>Buetow, L., Huang, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+catalysis+and+regulation+of+E3+ubiquitin+ligases.">Structural insights into the catalysis and regulation of E3 ubiquitin ligases.</a> Nature Reviews Molecular Cell Biology. 17 (10), 626-642 (2016).</li><li>French, M. E., Koehler, C. F., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+functions+of+branched+ubiquitin+chains.">Emerging functions of branched ubiquitin chains.</a> Cell Discovery. 7, 6 (2021).</li><li>Hatakeyama, S., Yada, M., Matsumoto, M., Ishida, N., Nakayama, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U+box+proteins+as+a+new+family+of+ubiquitin-protein+ligases.">U box proteins as a new family of ubiquitin-protein ligases.</a> Journal of Biological Chemistry. 276 (35), 33111-33120 (2001).</li><li>Herhaus, L., Dikic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+ubiquitin+code+through+post-translational+modification.">Expanding the ubiquitin code through post-translational modification.</a> EMBO Reports. 16 (9), 1071-1083 (2015).</li><li>Deshaies, R. J., Joazeiro, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RING+domain+E3+ubiquitin+ligases.">RING domain E3 ubiquitin ligases.</a> Annual Review of Biochemistry. 78, 399-434 (2009).</li><li>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=Lingering+mysteries+of+ubiquitin-chain+assembly.">Lingering mysteries of ubiquitin-chain assembly.</a> Cell. 124 (1), 27-34 (2006).</li><li>Hoppe, T., Cohen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organismal+protein+homeostasis+mechanisms.">Organismal protein homeostasis mechanisms.</a> Genetics. 215 (4), 889-901 (2020).</li><li>Okiyoneda, T., 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=Peripheral+protein+quality+control+removes+unfolded+CFTR+from+the+plasma+membrane.">Peripheral protein quality control removes unfolded CFTR from the plasma membrane.</a> Science. 329 (5993), 805-810 (1997).</li><li>Tawo, R., 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+Ubiquitin+ligase+CHIP+integrates+proteostasis+and+aging+by+regulation+of+insulin+receptor+turnover.">The Ubiquitin ligase CHIP integrates proteostasis and aging by regulation of insulin receptor turnover.</a> Cell. 169 (3), 470-482 (2017).</li><li>Albert, M. 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=CHIP+ubiquitylates+NOXA+and+induces+its+lysosomal+degradation+in+response+to+DNA+damage.">CHIP ubiquitylates NOXA and induces its lysosomal degradation in response to DNA damage.</a> Cell Death and Disease. 11 (9), 740 (2020).</li><li>Hoppe, T., 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=Regulation+of+the+myosin-directed+chaperone+UNC-45+by+a+novel+E3/E4-multiubiquitylation+complex+in+C.+elegans.">Regulation of the myosin-directed chaperone UNC-45 by a novel E3/E4-multiubiquitylation complex in C. elegans.</a> Cell. 118 (3), 337-349 (2004).</li><li>Kim, J., L&#246;we, T., Hoppe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+quality+control+gets+muscle+into+shape.">Protein quality control gets muscle into shape.</a> Trends in Cell Biology. 18 (6), 264-272 (2008).</li><li>Janiesch, P. 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=The+ubiquitin-selective+chaperone+CDC-48/p97+links+myosin+assembly+to+human+myopathy.">The ubiquitin-selective chaperone CDC-48/p97 links myosin assembly to human myopathy.</a> Nature Cell Biology. 9 (4), 379-390 (2007).</li><li>Donkervoort, 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=Pathogenic+variants+in+the+myosin+chaperone+UNC-45B+cause+progressive+myopathy+with+eccentric+cores.">Pathogenic variants in the myosin chaperone UNC-45B cause progressive myopathy with eccentric cores.</a> The American Journal of Human Genetics. 107 (6), 1078-1095 (2020).</li><li>Murata, S., Minami, Y., Minami, M., Chiba, T., Tanaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHIP+is+a+chaperone-dependent+E3+ligase+that+ubiquitylates+unfolded+protein.">CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein.</a> EMBO Reports. 2 (12), 1133-1138 (2001).</li><li>Jiang, 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=CHIP+is+a+U-box-dependent+E3+ubiquitin+ligase:+identification+of+Hsc70+as+a+target+for+ubiquitylation.">CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation.</a> Journal of Biological Chemistry. 276 (64), 42938-42944 (2001).</li><li>Yang, Y., Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+apoptosis:+the+ubiquitous+way.">Regulation of apoptosis: the ubiquitous way.</a> The FASEB Journal. 17 (8), 790-799 (2003).</li><li>Lamothe, B., 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=TRAF6+ubiquitin+ligase+is+essential+for+RANKL+signaling+and+osteoclast+differentiation.">TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation.</a> Biochemical and Biophysical Research Communication. 359 (4), 1044-1049 (2007).</li><li>Amemiya, Y., Azmi, P., Seth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoubiquitination+of+BCA2+RING+E3+ligase+regulates+its+own+stability+and+affects+cell+migration.">Autoubiquitination of BCA2 RING E3 ligase regulates its own stability and affects cell migration.</a> Molecular Cancer Research. 6 (9), 1385 (2008).</li><li>Brzovic, P. S., Lissounov, A., Christensen, D. E., Hoyt, D. W., Klevit, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+UbcH5/ubiquitin+noncovalent+complex+is+required+for+processive+BRCA1-directed+ubiquitination.">A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination.</a> Molecular Cell. 21 (6), 873-880 (2006).</li><li>Sakata, E., 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=Crystal+structure+of+UbcH5b~ubiquitin+intermediate:+Insight+into+the+formation+of+the+self-assembled+E2~Ub+conjugates.">Crystal structure of UbcH5b~ubiquitin intermediate: Insight into the formation of the self-assembled E2~Ub conjugates.</a> Structure. 18 (1), 138-147 (2010).</li><li>Eddins, M. J., Carlile, C. M., Gomez, K. M., Pickart, C. M., Wolberger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mms2-Ubc13+covalently+bound+to+ubiquitin+reveals+the+structural+basis+of+linkage-specific+polyubiquitin+chain+formation.">Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation.</a> Nature Structural and Molecular Biology. 13 (10), 915-920 (2006).</li><li>Buetow, L., 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=Activation+of+a+primed+RING+E3-E2+ubiquitin+complex+by+non-covalent+ubiquitin.">Activation of a primed RING E3-E2 ubiquitin complex by non-covalent ubiquitin.</a> Molecular Cell. 58 (2), 297-310 (2015).</li><li>Dou, H., Buetow, L., Sibbet, G. J., Cameron, K., Huang, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BIRC7-E2+ubiquitin+conjugate+structure+reveals+the+mechanism+of+ubiquitin+transfer+by+a+RING+dimer.">BIRC7-E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer.</a> Nature Structural and Molecular Biology. 19 (9), 876-883 (2012).</li><li>McKenna, 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=Noncovalent+interaction+between+ubiquitin+and+the+human+DNA+repair+protein+Mms2+is+required+for+Ubc13-mediated+polyubiquitination.">Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination.</a> Journal of Biological Chemistry. 276 (43), 40120-40125 (2001).</li><li>Plechanovov&#225;, 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=Mechanism+of+ubiquitylation+by+dimeric+RING+ligase+RNF4.">Mechanism of ubiquitylation by dimeric RING ligase RNF4.</a> Nature Structural Biology. 18 (9), 1052-1059 (2011).</li><li>Pierce, N. W., Kleiger, G., Shan, S. O., Deshaies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+sequential+polyubiquitylation+on+a+millisecond+timescale.">Detection of sequential polyubiquitylation on a millisecond timescale.</a> Nature. 462 (7273), 615-619 (2009).</li><li>Jain, A. K., Barton, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+p53:+TRIM24+enters+the+RING.">Regulation of p53: TRIM24 enters the RING.</a> Cell Cycle. 8 (22), 3668-3674 (2009).</li><li>Swatek, K. N., Komander, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+modifications.">Ubiquitin modifications.</a> Cell Research. 26, 399-422 (2016).</li><li>Yan, K., 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+role+of+K63-linked+polyubiquitination+in+cardiac+hypertrophy.">The role of K63-linked polyubiquitination in cardiac hypertrophy.</a> Journal of Cellular and Molecular Medicine. 22 (10), 4558-4567 (2018).</li><li>Dammer, E. B., 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=Polyubiquitin+linkage+profiles+in+three+models+of+proteolytic+stress+suggest+the+etiology+of+Alzheimer+disease.">Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease.</a> Journal of Biological Chemistry. 286 (12), 10457-10465 (2011).</li></ol>

本文介绍了用于分析E3连接酶功能的基本 体外 泛素化方法。在进行 体外 泛素化测定时，应考虑一些E2酶可以进行自泛素化，因为它们的活性半胱氨酸攻击其自身的赖氨酸残基，这些赖氨酸残基位于活性位点30附近。为了规避这个问题，建议使用E2突变体，其中将相应的赖氨酸残基交换为精氨酸，这导致催化活性E2酶对自身泛素化32具有抗性。当通过...

泛素化是Ub与底物蛋白1共价连接的过程。Ub修饰由连续的酶促反应催化，涉及三种不同酶类的作用，即Ub激活酶（E1s），Ub偶联酶（E2s），Ub连接酶（E3s）和可能的Ub链伸长因子（E4s）2，3，4，5。在三磷酸腺苷（ATP）和镁（Mg2+）依赖性Ub被E1激活后，E1的活性位点半胱氨酸攻击Ub的C端甘氨酸，形成硫酯复合物（Ub~E1）。从ATP水解中汲取的能量使Ub达到高能量过渡状态，该状态在随后的酶级联中保持。接下来，E2酶将活化的Ub转移到其内部催化半胱氨酸中，从而形成瞬态Ub~E2硫酯键。随后，Ub被转移到底物蛋白上。
这可以通过两种方式完成。E3连接酶可以首先与E2结合，或者E3连接酶可以直接结合Ub。后一种方式导致E3~Ub中间体的形成。在任何一种情况下，Ub通过在Ub的C端羧基和底物6的赖氨酸Ɛ-氨基之间形成同肽键与底物蛋白连接。人类基因组编码两个E1，大约40个E2和600多个推定的泛素连接酶7。基于E3的Ub转移机制，将Ub连接酶分为三类，涉及E6AP C-Terminus（HECT）型同源，真正有趣的新基因（RING）/U-box型，以及RING（RBR）型连接酶8之间的RING。在这项研究中，含有连接酶的U盒，HSC70相互作用蛋白（CHIP）的羧基终点，被用作代表性的E3酶。与形成Ub~E3硫代酯的HECT型E3酶相反，CHIP的U-box结构域结合E2~Ub并促进随后的Ub/底物直接从E2酶8，9转移。基于U型箱对酶促功能的重要性，使用非活性的芯片U型箱突变体CHIP（H260Q）作为对照。CHIP（H260Q）无法与其同源E2s结合，从而失去其E3连接酶活性10。
蛋白质泛素化在调节真核细胞中的多种细胞事件中起着至关重要的作用。Ub分子与底物蛋白的可逆附着促进的细胞结果的多样性可归因于Ub的分子特性。由于Ub本身含有七个赖氨酸（K）残基，用于进一步泛素化，因此存在丰富多样的具有不同尺寸和/或拓扑结构的Ub链类型11。例如，底物可以被单个Ub分子在一个（单泛素化）或多个赖氨酸（多单泛素化）修饰，甚至通过Ub链（多泛素化）11。Ub链通过相同或不同的Ub赖氨酸残基形成同型或异型，这甚至可能导致支链Ub链9。因此，蛋白质泛素化导致Ub分子的多种排列，提供特异性信息，例如，用于偶联蛋白的降解，活化或定位12，13。这些不同的Ub信号能够快速重编程细胞信号通路，这是细胞响应不断变化的环境需求能力的重要要求。
泛素化的一个核心方面与蛋白质质量控制有关。错误折叠或不可逆受损的蛋白质必须被降解并被新合成的蛋白质取代，以维持蛋白质稳态或蛋白质稳态14。质量控制E3连接酶，CHIP，与分子伴侣合作，在受损蛋白9，15，16，17的Ub依赖性降解中合作。除此之外，CHIP调节肌球素导向的伴侣UNC-45B（Unc-45同源物B）的稳定性，其与肌肉功能紧密协调，偏离最佳水平导致人肌病18，19，20，21。26S蛋白酶体对UNC-45B的降解是由K48连接的聚Ub链9的附着介导的。在没有底物蛋白的情况下，CHIP进行自泛素化10，22，23，这是RING/ U-box E3泛素连接酶24，25的特征，并被认为调节连接酶活性26。本文中描述的体外泛素化测定方法的应用有助于系统地鉴定与CHIP合作促进游离聚Ub链形成和/或CHIP的自动泛素化的E2酶（方案部分2）。此外，观察到UNC-45B的CHIP依赖性泛素化，这是E3连接酶18，19的已知底物（方案部分3）。最终，监测了来自Ub~E2硫酯的活化Ub的CHIP依赖性转移（协议第4节）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amershan Protran 0.1 &#181;m NC</td>
			<td>GE Healthcare</td>
			<td>10600000</td>
			<td>nitrocellulose membrane</td>
		</tr>
		<tr>
			<td>Anti-CHIP</td>
			<td>Cell Signaling</td>
			<td>2080</td>
			<td>Monoclonal rabbit anti-CHIP antibody, clone C3B6</td>
		</tr>
		<tr>
			<td>Anti-MYC</td>
			<td>Roche</td>
			<td>OP10</td>
			<td>Monoclonal mouse anti-MYC antibody, clone 9E10</td>
		</tr>
		<tr>
			<td>Anti-ubiquitin</td>
			<td>Upstate</td>
			<td>05-944</td>
			<td>Monoclonal mouse anti-Ub antibody, clone P4D1-A11</td>
		</tr>
		<tr>
			<td>Apyrase</td>
			<td>Sigma</td>
			<td>A6535-100UN</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP (10x)</td>
			<td>Enzo</td>
			<td>12091903</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A6003-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Roth</td>
			<td>P749.2</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Roth</td>
			<td>3904.1</td>
			<td></td>
		</tr>
		<tr>
			<td>LDS sample buffer (4x)</td>
			<td>novex</td>
			<td>B0007</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma</td>
			<td>L5501-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Roth</td>
			<td>4256.4</td>
			<td></td>
		</tr>
		<tr>
			<td>MeOH</td>
			<td>VWR Chemicals</td>
			<td>2,08,47,307</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>Milchpulver</td>
			<td>Roth</td>
			<td>T145.3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Roth</td>
			<td>P029.3</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Antioxidant</td>
			<td>invitrogen</td>
			<td>NP0005</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Transfer buffer (20x)</td>
			<td>novex</td>
			<td>NP0006-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Page ruler plus</td>
			<td>Thermo Fisher</td>
			<td>26619</td>
			<td>Protein ladder</td>
		</tr>
		<tr>
			<td>RotiBlock</td>
			<td>Roth</td>
			<td>A151.1</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>SDS (20%)</td>
			<td>Roth</td>
			<td>1057.1</td>
			<td></td>
		</tr>
		<tr>
			<td>S1000 Thermal Cycler</td>
			<td>Bio Rad</td>
			<td>1852196</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo</td>
			<td>Bio Rad</td>
			<td>1704150EDU</td>
			<td>Transfer system</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Roth</td>
			<td>4855.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Roth</td>
			<td>9127.2</td>
			<td></td>
		</tr>
		<tr>
			<td>UbcH Enzyme Set</td>
			<td>BostonBiochem</td>
			<td>K-980B</td>
			<td>E2 enzymes</td>
		</tr>
		<tr>
			<td>Ubiquitin</td>
			<td>BostonBiochem</td>
			<td>U-100H</td>
			<td></td>
		</tr>
		<tr>
			<td>Ubiquitin-activating enzyme E1</td>
			<td>Enzo</td>
			<td>BML-UW941U-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Ubiquitylation buffer (10x)</td>
			<td>Enzo</td>
			<td>BML-KW9885-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman blotting paper</td>
			<td>Bio Rad</td>
			<td>1703969</td>
			<td>Extra thick filter paper</td>
		</tr>
	</tbody>
</table>

in vitro analysis of e3 ubiquitin ligase function

泛素（Ub）与底物蛋白的内部赖氨酸残基的共价附着，这一过程称为泛素化，代表了真核生物中最重要的翻译后修饰之一。泛素化由三种酶类的顺序级联介导，包括泛素激活酶（E1酶），泛素偶联酶（E2酶）和泛素连接酶（E3酶），有时还有泛素链伸长因子（E4酶）。在这里，提供了用于泛素化测定 的体外 方案，允许评估E3泛素连接酶活性，E2-E3对之间的合作以及底物选择。可以通过监测游离多泛素链的产生和/或E3连接酶的自动泛素化来筛选合作的E2-E3对。底物泛素化通过E3连接酶的选择性结合来定义，并且可以通过 体外 反应的蛋白质印迹来检测。此外，还描述了E2~Ub放电测定法，这是直接评估功能E2-E3合作的有用工具。在这里，泛素的E3依赖性转移从相应的E2酶跟随到游离赖氨酸氨基酸（模仿底物泛素化）或E3连接酶本身的内部赖氨酸（自泛素化）。总之，提供了三种不同的 体外 方案，这些方案快速且易于执行，以解决E3连接酶催化功能。

为了鉴定与泛素连接酶CHIP合作的E2酶，在单个 体外 泛素化反应中测试了一组E2候选酶。通过E3依赖性泛素化产物的形成来监测配合的E2-E3对， 即E3连接酶的自泛素化和游离Ub聚合物的形成。通过蛋白质印迹法分析泛素化产物。数据解释基于所得蛋白质条带与分子量标记物的大小比较。蛋白质泛素化导致特定能带模式的形成，其特征在于出现双条带或多个迭代带，其大小差分别为8.6 kDa?...

Watch this Scientific Journal Video about In Vitro Analysis of E3 Ubiquitin Ligase Function at JoVE.com

In Vitro Analysis of E3 Ubiquitin Ligase Function

本研究为分析E3泛素连接酶催化活性提供了详细的 体外 泛素化测定方案。重组蛋白使用原核系统（如 大肠 杆菌培养物）表达。

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

该协议的总体目标是分析E3连接酶功能的重要关键方面，包括E2酶特异性，底物选择和泛素转移。该技术的主要优点是其广泛的适用性，因为来自所有真核来源的蛋白质可以在体外重组表达和研究，而无需特殊设备。首次使用赖氨酸放电技术时，重要的是优化测定参数，例如酶浓度，以确定所选E3连接酶的理想放电条件。为了进行体外自动泛素化测定，建立移液方案以测试不同E2酶的功能能力，并在冰上制备用于所有泛素化反应和一个额外反应的预混液。接下来，将一微摩尔E2酶加入适当的试管中，并在指定的条件下将样品在PCR热循环仪中孵育两小时。孵育后，向每个反应中加入SDS样品缓冲液，并通过移液多次混合。然后立即将样品在95摄氏度下煮沸五分钟，并将变性蛋白质储存在20摄氏度。为了进行体外底物泛素化测定，请为所有反应准备移液方案。在计算一次反应所需的预混液量后，在冰上制备所有反应的预混液，并将预混液加入每个管中。单独添加相应的 E3 连接酶。然后如图所示，将样品在PCR热循环仪中孵育两小时。在孵育结束时，向每个反应中加入两次SDS样品缓冲液，通过移液混合几次，并在95摄氏度下将样品煮沸五分钟。要进行赖氨酸放电测定，请设置装药反应的移液方案，并将装药反应在37摄氏度下孵育15分钟。为了停止充电反应，将烧焦酶加入到每毫升1.8单位的终浓度中，并在室温下孵育反应五分钟。在孵育结束时，将EDTA加入至终浓度为30毫摩尔，并使用双蒸馏水将反应体积调节至30微升。为了通过E3放电E2泛素化，设置五个对应于实验时间点的管，向每个管中加入6.7微升非还原性样品缓冲液，从时间点零管中除去6微升停止充电反应，并将总体积调节至26.7微升。为了建立放电反应，向每个反应中加入双蒸馏水，泛素化缓冲液，BSA，赖氨酸，E3连接酶和带电的E2。在选定的时间段后，将20微升样品从每次放电反应转移到相应的样品管中。然后立即涡旋样品，并将它们置于70摄氏度下10分钟。在这项具有代表性的蛋白质印迹分析中，非活性芯片不是自动泛素化的。然而，E3非依赖性的泛素产物是在存在非活性CHIP和没有CHIP的情况下形成的。野生型CHIP与UBE2D和UBE2E家族的蛋白质结合时是自泛素化的，而游离的多泛素链是与UBE2D家族的蛋白质合作生产的，而不是与UBE2E家族的蛋白质合作生产的。UBE2N/V1对泛素的结合引导游离泛素链的形成。使用野生型CHIP观察到UNC-45B的自泛素化和泛素化，但未观察到CHIP的非活性突变体，这表明UNC-45B充当CHIP的保守底物。当使用赖氨酸放电测定法分析CHIP的催化活性时，未带电的UBE2D2酶的分子量为17千道尔顿，具有单个泛素分子的带电UBE2D2的分子量约为26千道尔顿。在时间 0 处，观察到整个带电的 E2 当量。在存在非活性CHIP的情况下，检测到UBE2D2的微弱E3连接酶非依赖性放电，但未检测到CHIP的自泛素化。在野生型CHIP存在下，UBE2D2的放电速度快，在60分钟内完成，CHIP的自泛素化表明泛素转移到其自身赖氨酸残基上。由于E2能力有限，请尽快工作并立即进行放电反应，然后进行SDS-PAGE和蛋白质印迹。遵循这些体外泛素化方案，可以通过用连锁特异性抗体探测蛋白质印迹来鉴定特定的泛素化连锁类型。

Research

JoVE Journal

Biology

In Vitro Analysis of E3 Ubiquitin Ligase Function

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

科研

生物学

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

依赖的PDZ-CFTR的大分子信号复合物的体外分析

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

基因功能分析和可视化的纤毛产生的流体流动的在枯否囊泡中

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

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 ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

肾单位的组成与功能的成年斑马鱼肾脏分析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...