用蓝色 native 电泳法估算天然蛋白质的分子量：原理及实用提示。
Mass estimation of native proteins by blue native electrophoresis: principles and practical hints.
机构信息
Molecular Bioenergetics, Medical School, Goethe-Universität Frankfurt, Theodor-Stern-Kai 7, Haus 26, D-60590 Frankfurt am Main, Germany.
出版信息
Mol Cell Proteomics. 2010 Oct;9(10):2149-61. doi: 10.1074/mcp.M900526-MCP200. Epub 2010 Feb 20.
Abstract
Blue native electrophoresis is one of the most popular techniques for mass estimation of native membrane proteins, but the use of non-optimal mass markers and acrylamide gels can compromise accuracy and reliability of the results. We present short protocols taking 10-30 min to prepare optimal sets of membrane protein markers from chicken, rat, mouse, and bovine heart. Especially heart materials from local supermarkets or butcher's shops, e.g. chicken or bovine heart, are ideal sources of high mass membrane protein standards. Considerable discrepancies between the migration behavior of membrane and soluble markers suggest using membrane protein markers for mass estimation of membrane proteins. Soluble standard proteins can be used, with some limitations, when soluble proteins are the focus. Principles and general rules for the determination of mass and oligomeric state of native membrane and soluble proteins are elaborated, and potential pitfalls are discussed.
摘要
蓝色 native 电泳是一种最受欢迎的用于大规模估计天然膜蛋白的技术，但使用非最佳的质量标记物和丙烯酰胺凝胶会影响结果的准确性和可靠性。我们提供了简短的方案，这些方案只需 10-30 分钟即可从鸡、鼠、牛心脏中制备最佳的膜蛋白标记物。特别是来自当地超市或肉店的心脏材料，如鸡或牛心，是高质量膜蛋白标准的理想来源。膜和可溶性标记物的迁移行为之间存在很大差异，这表明使用膜蛋白标记物来估计膜蛋白的质量。在关注可溶性蛋白时，可以使用可溶性标准蛋白，但存在一些限制。详细阐述了确定天然膜和可溶性蛋白的质量和寡聚状态的原则和一般规则，并讨论了潜在的陷阱。
相似文献
1
Mass estimation of native proteins by blue native electrophoresis: principles and practical hints.用蓝色 native 电泳法估算天然蛋白质的分子量：原理及实用提示。
Mol Cell Proteomics. 2010 Oct;9(10):2149-61. doi: 10.1074/mcp.M900526-MCP200. Epub 2010 Feb 20.
2
Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.通过蓝色非变性电泳分析蛋白质复合物的分子量和寡聚状态，以及通过二维非变性电泳分离膜蛋白复合物。
Anal Biochem. 1994 Mar;217(2):220-30. doi: 10.1006/abio.1994.1112.
3
LC-nanospray-MS/MS analysis of hydrophobic proteins from membrane protein complexes isolated by blue-native electrophoresis.通过蓝色非变性电泳分离的膜蛋白复合物中疏水蛋白的液相色谱-纳喷雾串联质谱分析。
J Mass Spectrom. 2005 Sep;40(9):1223-31. doi: 10.1002/jms.903.
4
A cost-effective method for simultaneous homo-oligomeric size determination and monodispersity conditions for membrane proteins.一种用于同时测定同源寡聚体大小和膜蛋白单分散性条件的经济有效方法。
Anal Biochem. 2011 Sep 1;416(1):100-6. doi: 10.1016/j.ab.2011.05.007. Epub 2011 May 12.
5
Separation of proteins by blue native electrophoresis.通过蓝色非变性电泳分离蛋白质。
Methods Mol Biol. 2007;390:417-27. doi: 10.1007/978-1-59745-466-7_28.
6
Isolation of membrane protein complexes by blue native electrophoresis.通过蓝色非变性电泳分离膜蛋白复合物
Methods Mol Biol. 2008;424:423-31. doi: 10.1007/978-1-60327-064-9_33.
7
Analysis of membrane protein complexes by blue native PAGE.通过蓝色天然聚丙烯酰胺凝胶电泳分析膜蛋白复合物
Proteomics. 2006 Sep;6 Suppl 2:6-15. doi: 10.1002/pmic.200600553.
8
Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation.用蓝色非变性电泳和分析型超速离心法分析膜转运蛋白的寡聚状态。
J Mol Biol. 2002 Apr 5;317(4):591-600. doi: 10.1006/jmbi.2002.5416.
9
Advantages and limitations of clear-native PAGE.清晰天然聚丙烯酰胺凝胶电泳的优点和局限性
Proteomics. 2005 Nov;5(17):4338-46. doi: 10.1002/pmic.200500081.
10
Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts.丙烯酰胺浓度决定螺旋膜蛋白凝胶迁移的方向和幅度。
Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15668-73. doi: 10.1073/pnas.1311305110. Epub 2013 Sep 9.
引用本文的文献
1
Recent advances in the structure, function and regulation of the volume-regulated anion channels and their role in immunity.容积调节性阴离子通道的结构、功能、调节及其在免疫中的作用的最新进展
J Physiol. 2024 Dec 22. doi: 10.1113/JP285200.
2
Insights into Transient Dimerisation of Carnitine/Acylcarnitine Carrier (SLC25A20) from Sarkosyl/PAGE, Cross-Linking Reagents, and Comparative Modelling Analysis.从 Sarkosyl/PAGE、交联试剂和比较建模分析中洞察肉碱/酰基辅酶 A 载体（SLC25A20）的瞬时二聚化。
Biomolecules. 2024 Sep 14;14(9):1158. doi: 10.3390/biom14091158.
3
Analysis of Complexome Profiles with the Gaussian Interaction Profiler (GIP) Reveals Novel Protein Complexes in .用高斯交互分析器（GIP）分析复杂蛋白质组图谱揭示了 中的新型蛋白质复合物。
J Proteome Res. 2024 Oct 4;23(10):4467-4479. doi: 10.1021/acs.jproteome.4c00414. Epub 2024 Sep 12.
4
OXPHOS Organization and Activity in Mitochondria of Plants with Different Life Strategies.不同生活策略植物线粒体中的 OXPHOS 组织和活性。
Int J Mol Sci. 2023 Oct 16;24(20):15229. doi: 10.3390/ijms242015229.
5
Reconstitution of a minimal ESX-5 type VII secretion system suggests a role for PPE proteins in the outer membrane transport of proteins.重建最小的 ESX-5 型 VII 分泌系统表明 PPE 蛋白在蛋白质的外膜转运中起作用。
mSphere. 2023 Oct 24;8(5):e0040223. doi: 10.1128/msphere.00402-23. Epub 2023 Sep 25.
6
Let's make it clear: systematic exploration of mitochondrial DNA- and RNA-protein complexes by complexome profiling.让我们明确一点：通过复合物组分析对线粒体DNA和RNA-蛋白质复合物进行系统探索。
Nucleic Acids Res. 2023 Oct 27;51(19):10619-10641. doi: 10.1093/nar/gkad697.
7
Mitochondrial complexome reveals quality-control pathways of protein import.线粒体复合物组揭示了蛋白质输入的质量控制途径。
Nature. 2023 Feb;614(7946):153-159. doi: 10.1038/s41586-022-05641-w. Epub 2023 Jan 25.
8
Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1.植物 NRC 类辅助 NLR 通过感应 NLR 免疫受体 Rpi-amr3 和 Rpi-amr1 的效应子依赖激活和寡聚化。
EMBO J. 2023 Mar 1;42(5):e111484. doi: 10.15252/embj.2022111484. Epub 2023 Jan 2.
9
Separation and analysis of Bacillus subtilis respiratory chain complexes.枯草芽孢杆菌呼吸链复合物的分离与分析。
J Bioenerg Biomembr. 2022 Dec;54(5-6):251-271. doi: 10.1007/s10863-022-09951-6. Epub 2022 Nov 23.
10
Arginine 125 Is an Essential Residue for the Function of MRAP2.精氨酸 125 是 MRAP2 功能所必需的关键残基。
Int J Mol Sci. 2022 Aug 30;23(17):9853. doi: 10.3390/ijms23179853.
本文引用的文献
1
Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes.线粒体膜中ATP合酶与呼吸链的超分子组织
Biochim Biophys Acta. 2009 Jun;1787(6):672-80. doi: 10.1016/j.bbabio.2008.12.016. Epub 2009 Jan 8.
2
Proton-translocating transhydrogenase: an update of unsolved and controversial issues.质子转运型转氢酶：未解决及有争议问题的最新进展
J Bioenerg Biomembr. 2008 Oct;40(5):463-73. doi: 10.1007/s10863-008-9170-x. Epub 2008 Oct 30.
3
Features and applications of blue-native and clear-native electrophoresis.蓝色非变性和清亮非变性电泳的特点与应用
Proteomics. 2008 Oct;8(19):3974-90. doi: 10.1002/pmic.200800017.
4
The ADP and ATP transport in mitochondria and its carrier.线粒体中的二磷酸腺苷（ADP）和三磷酸腺苷（ATP）转运及其载体
Biochim Biophys Acta. 2008 Oct;1778(10):1978-2021. doi: 10.1016/j.bbamem.2008.04.011. Epub 2008 May 2.
5
Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.在高分辨率清晰天然凝胶中进行功能分析，以量化人类活检组织和细胞系中的线粒体复合物。
Electrophoresis. 2007 Nov;28(21):3811-20. doi: 10.1002/elps.200700367.
6
Identification of two proteins associated with mammalian ATP synthase.与哺乳动物ATP合酶相关的两种蛋白质的鉴定。
Mol Cell Proteomics. 2007 Oct;6(10):1690-9. doi: 10.1074/mcp.M700097-MCP200. Epub 2007 Jun 17.
7
High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.用于膜蛋白复合物凝胶内功能测定和荧光研究的高分辨率清晰天然电泳。
Mol Cell Proteomics. 2007 Jul;6(7):1215-25. doi: 10.1074/mcp.M700076-MCP200. Epub 2007 Apr 9.
8
Blue native PAGE.蓝色非变性聚丙烯酰胺凝胶电泳
Nat Protoc. 2006;1(1):418-28. doi: 10.1038/nprot.2006.62.
9
Energy transduction: proton transfer through the respiratory complexes.能量转导：质子通过呼吸复合体的转移。
Annu Rev Biochem. 2006;75:165-87. doi: 10.1146/annurev.biochem.75.062003.101730.
10
Energy converting NADH:quinone oxidoreductase (complex I).能量转换型NADH：醌氧化还原酶（复合体I）。
Annu Rev Biochem. 2006;75:69-92. doi: 10.1146/annurev.biochem.75.103004.142539.
Zotero 插件