从人细胞中对带有TAP标签的功能性蛋白质进行串联亲和纯化。
Tandem affinity purification of functional TAP-tagged proteins from human cells.
作者信息
Gregan Juraj, Riedel Christian G, Petronczki Mark, Cipak Lubos, Rumpf Cornelia, Poser Ina, Buchholz Frank, Mechtler Karl, Nasmyth Kim
机构信息
Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Dr Bohr-Gasse 1, 1030 Vienna, Austria.
出版信息
Nat Protoc. 2007;2(5):1145-51. doi: 10.1038/nprot.2007.172.
Abstract
Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.
摘要
串联亲和纯化（TAP）是一种通用的两步亲和纯化方案，用于分离带有TAP标签的蛋白质及其相关蛋白。我们使用细菌人工染色体在人HeLa细胞中异源表达带有TAP标签的小鼠Sgo1蛋白。这使我们能够通过RNA干扰介导的内源性人Sgo1缺失来测试Sgo1-TAP蛋白的功能。在此，我们提供了一种从HeLa细胞中纯化带有TAP标签的Sgo1蛋白以及KIAA1387的优化方案，并附有详细说明。该纯化方案可在1天内完成，且应适用于其他蛋白质。
相似文献
1
Tandem affinity purification of functional TAP-tagged proteins from human cells.从人细胞中对带有TAP标签的功能性蛋白质进行串联亲和纯化。
Nat Protoc. 2007;2(5):1145-51. doi: 10.1038/nprot.2007.172.
2
Tandem affinity purification of protein complexes from mammalian cells by the Strep/FLAG (SF)-TAP tag.利用链霉亲和素/FLAG（SF）串联亲和纯化标签从哺乳动物细胞中纯化蛋白质复合物。
Methods Mol Biol. 2009;564:359-72. doi: 10.1007/978-1-60761-157-8_21.
3
Strep/FLAG tandem affinity purification (SF-TAP) to study protein interactions.用于研究蛋白质相互作用的链霉亲和素/FLAG串联亲和纯化（SF-TAP）。
Curr Protoc Protein Sci. 2009 Aug;Chapter 19:19.20.1-19.20.19. doi: 10.1002/0471140864.ps1920s57.
4
A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes.一种用于高效分离和鉴定天然蛋白质复合物的新型串联亲和纯化策略。
Proteomics. 2007 Dec;7(23):4228-34. doi: 10.1002/pmic.200700038.
5
Interaction proteomics: characterization of protein complexes using tandem affinity purification-mass spectrometry.相互作用蛋白质组学：使用串联亲和纯化-质谱法对蛋白质复合物进行表征。
Biochem Soc Trans. 2010 Aug;38(4):883-7. doi: 10.1042/BST0380883.
6
Tandem affinity purification of ciliopathy-associated protein complexes.纤毛病相关蛋白复合物的串联亲和纯化
Methods Cell Biol. 2009;91:143-60. doi: 10.1016/S0091-679X(08)91009-8. Epub 2009 Dec 1.
7
An efficient protein complex purification method for functional proteomics in higher eukaryotes.一种用于高等真核生物功能蛋白质组学的高效蛋白质复合物纯化方法。
Nat Biotechnol. 2003 Jan;21(1):89-92. doi: 10.1038/nbt773. Epub 2002 Dec 16.
8
Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris.用于毕赤酵母内源性蛋白质复合物的六组氨酸-3×FLAG串联亲和纯化方法的开发。
J Struct Funct Genomics. 2014 Dec;15(4):191-9. doi: 10.1007/s10969-014-9190-1. Epub 2014 Nov 15.
9
Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.从真核细胞中进行蛋白质复合物的串联亲和纯化
J Vis Exp. 2017 Jan 26(119):55236. doi: 10.3791/55236.
10
Sequential peptide affinity purification system for the systematic isolation and identification of protein complexes from Escherichia coli.用于从大肠杆菌中系统分离和鉴定蛋白质复合物的连续肽亲和纯化系统。
Methods Mol Biol. 2009;564:373-400. doi: 10.1007/978-1-60761-157-8_22.
引用本文的文献
1
Protocol to profile spatially resolved NLRP3 inflammasome complexes using APEX2-based proximity labeling.使用基于APEX2的邻近标记对空间分辨的NLRP3炎性小体复合物进行分析的方案。
STAR Protoc. 2024 Dec 20;5(4):103417. doi: 10.1016/j.xpro.2024.103417. Epub 2024 Oct 25.
2
Progress in mass spectrometry approaches to profiling protein-protein interactions in the studies of the innate immune system.质谱分析法在天然免疫系统研究中用于分析蛋白质-蛋白质相互作用的进展。
J Proteins Proteom. 2024 Sep;15(3):545-559. doi: 10.1007/s42485-024-00156-6. Epub 2024 Jun 28.
3
Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins.串联亲和纯化和质谱分析 FACT 及其相关蛋白。
Methods Mol Biol. 2023;2701:209-227. doi: 10.1007/978-1-0716-3373-1_14.
4
Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons.通过串联亲和纯化-质谱法鉴定原代神经元中Kv4.2蛋白复合物及其修饰
Front Cell Neurosci. 2022 Dec 9;16:1070305. doi: 10.3389/fncel.2022.1070305. eCollection 2022.
5
The Galaxy Platform for Reproducible Affinity Proteomic Mass Spectrometry Data Analysis.用于可重复亲和蛋白质组质谱数据分析的银河平台。
Methods Mol Biol. 2019;1977:249-261. doi: 10.1007/978-1-4939-9232-4_16.
6
Genetic selection for mistranslation rescues a defective co-chaperone in yeast.针对错译的基因选择挽救了酵母中一种有缺陷的共伴侣蛋白。
Nucleic Acids Res. 2017 Apr 7;45(6):3407-3421. doi: 10.1093/nar/gkw1021.
7
Identification of Novel Proteins Co-Purifying with Cockayne Syndrome Group B (CSB) Reveals Potential Roles for CSB in RNA Metabolism and Chromatin Dynamics.与科凯恩综合征B组（CSB）共同纯化的新型蛋白质的鉴定揭示了CSB在RNA代谢和染色质动力学中的潜在作用。
PLoS One. 2015 Jun 1;10(6):e0128558. doi: 10.1371/journal.pone.0128558. eCollection 2015.
8
SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs.SNW1通过介导sororin和APC2前体mRNA的剪接来实现姐妹染色单体黏连。
EMBO J. 2014 Nov 18;33(22):2643-58. doi: 10.15252/embj.201488202. Epub 2014 Sep 25.
9
The cockayne syndrome B protein is essential for neuronal differentiation and neuritogenesis.科凯恩综合征B蛋白对于神经元分化和神经突形成至关重要。
Cell Death Dis. 2014 May 29;5(5):e1268. doi: 10.1038/cddis.2014.228.
10
Optimisation of Downscaled Tandem Affinity Purifications to Identify Core Protein Complexes.优化缩减规模的串联亲和纯化以鉴定核心蛋白复合物
J Integr OMICS. 2012 May;2(1):55-68. doi: 10.5584/jiomics.v2i1.81.
本文引用的文献
1
Tricine-SDS-PAGE.三羟甲基氨基甲烷-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳
Nat Protoc. 2006;1(1):16-22. doi: 10.1038/nprot.2006.4.
2
An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.一种用于哺乳动物细胞相互作用蛋白质组学的高效串联亲和纯化方法。
Nat Methods. 2006 Dec;3(12):1013-9. doi: 10.1038/nmeth968. Epub 2006 Oct 22.
3
Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.酿酒酵母中蛋白质复合物的全球格局。
Nature. 2006 Mar 30;440(7084):637-43. doi: 10.1038/nature04670. Epub 2006 Mar 22.
4
Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I.蛋白磷酸酶2A在减数分裂I期间保护着丝粒姐妹染色单体黏连。
Nature. 2006 May 4;441(7089):53-61. doi: 10.1038/nature04664. Epub 2006 Mar 15.
5
Proteome survey reveals modularity of the yeast cell machinery.蛋白质组研究揭示酵母细胞机制的模块化特性。
Nature. 2006 Mar 30;440(7084):631-6. doi: 10.1038/nature04532. Epub 2006 Jan 22.
6
Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells.在脊椎动物细胞的有丝分裂过程中，守护蛋白可防止黏连蛋白从着丝粒上解离。
PLoS Biol. 2005 Mar;3(3):e86. doi: 10.1371/journal.pbio.0030086. Epub 2005 Mar 1.
7
Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting Shugoshin localization.人类Bub1通过影响守护蛋白的定位来确定有丝分裂染色体上的持续黏连位点。
Curr Biol. 2005 Feb 22;15(4):353-9. doi: 10.1016/j.cub.2004.12.044.
8
RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells.通过细菌人工染色体转基因在哺乳动物组织培养细胞中进行RNA干扰拯救。
Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2396-401. doi: 10.1073/pnas.0409861102. Epub 2005 Feb 3.
9
Interaction network containing conserved and essential protein complexes in Escherichia coli.包含大肠杆菌中保守且必需蛋白质复合物的相互作用网络。
Nature. 2005 Feb 3;433(7025):531-7. doi: 10.1038/nature03239.
10
A combined approach for the localization and tandem affinity purification of protein complexes from metazoans.一种用于后生动物蛋白质复合物定位和串联亲和纯化的联合方法。
Sci STKE. 2005 Jan 11;2005(266):pl1. doi: 10.1126/stke.2662005pl1.
Zotero 插件