一种用于测定溶液中游离蛋白质 - DNA 结合亲和力的毛细管电泳迁移率变动分析方法。
A capillary electrophoresis mobility shift assay for protein-DNA binding affinities free in solution.
作者信息
Foulds G J, Etzkorn F A
机构信息
Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, VA 22901, USA.
出版信息
Nucleic Acids Res. 1998 Sep 15;26(18):4304-5. doi: 10.1093/nar/26.18.4304.
Abstract
Quantitative determination of dissociation constants for DNA-protein complexes will help clarify the molecular mechanisms of transcription, replication and DNA repair. A practical capillary electrophoresis mobility shift assay (CEMSA) for protein-DNA affinities free in solution is presented. The method is fast and simple, precise and general. The speed (<2 min separations) and simplicity derive from the use of an uncoated capillary with no gel matrix. The dissociation constant for GCNK58, a DNA-binding-region construct of the yeast transcription factor GCN4, binding to the AP1 DNA site was measured ( K d = 35 +/- 4 nM) to demonstrate the utility of the method.
摘要
DNA-蛋白质复合物解离常数的定量测定将有助于阐明转录、复制和DNA修复的分子机制。本文介绍了一种用于测定溶液中蛋白质-DNA亲和力的实用毛细管电泳迁移率变动分析方法（CEMSA）。该方法快速简便、精确通用。其速度（分离时间<2分钟）和简便性源于使用无凝胶基质的未涂层毛细管。通过测定酵母转录因子GCN4的DNA结合区域构建体GCNK58与AP1 DNA位点的结合解离常数（Kd = 35 +/- 4 nM）来证明该方法的实用性。
相似文献
1
A capillary electrophoresis mobility shift assay for protein-DNA binding affinities free in solution.一种用于测定溶液中游离蛋白质 - DNA 结合亲和力的毛细管电泳迁移率变动分析方法。
Nucleic Acids Res. 1998 Sep 15;26(18):4304-5. doi: 10.1093/nar/26.18.4304.
2
Interaction of a bZip oligopeptide model with oligodeoxyribonucleotides modelling DNA binding sites. The effect of flanking sequences.一种bZip寡肽模型与模拟DNA结合位点的寡脱氧核糖核苷酸之间的相互作用。侧翼序列的影响。
J Biomol Struct Dyn. 1997 Dec;15(3):587-96. doi: 10.1080/07391102.1997.10508968.
3
Effects of phosphate neutralization on the shape of the AP-1 transcription factor binding site in duplex DNA.磷酸盐中和对双链DNA中AP-1转录因子结合位点形状的影响。
Nucleic Acids Res. 1998 May 15;26(10):2298-305. doi: 10.1093/nar/26.10.2298.
4
DNA recognition by peptide oligomers.肽寡聚物对DNA的识别
Nucleic Acids Symp Ser. 1995(34):143-4.
5
6
Coupled folding and site-specific binding of the GCN4-bZIP transcription factor to the AP-1 and ATF/CREB DNA sites studied by microcalorimetry.通过微量热法研究GCN4-bZIP转录因子与AP-1和ATF/CREB DNA位点的耦合折叠和位点特异性结合。
Biochemistry. 1996 Nov 26;35(47):14984-91. doi: 10.1021/bi961312a.
7
Kinetic studies of sequence-specific binding of GCN4-bZIP peptides to DNA strands immobilized on a 27-MHz quartz-crystal microbalance.GCN4-bZIP肽与固定在27兆赫兹石英晶体微天平上的DNA链的序列特异性结合的动力学研究。
Biochemistry. 1998 Apr 21;37(16):5666-72. doi: 10.1021/bi980037k.
8
Short, hydrophobic, alanine-based proteins based on the basic region/leucine zipper protein motif: overcoming inclusion body formation and protein aggregation during overexpression, purification, and renaturation.基于碱性区域/亮氨酸拉链蛋白基序的短链、疏水、基于丙氨酸的蛋白质：克服过表达、纯化和复性过程中的包涵体形成和蛋白质聚集。
Protein Expr Purif. 2000 Apr;18(3):394-403. doi: 10.1006/prep.2000.1209.
9
A general strategy to determine a target DNA sequence of a short peptide: application to a d-peptide.
J Am Chem Soc. 2002 Jan 16;124(2):180-1. doi: 10.1021/ja017078f.
10
Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization.酵母MATalpha2/MCM1/DNA三元复合物的结晶：蛋白质/DNA共结晶的一般方法和原理
J Mol Biol. 2000 Apr 7;297(4):947-59. doi: 10.1006/jmbi.2000.3606.
引用本文的文献
1
Versatile Analysis of DNA-Biomolecule Interactions in Solution by Hydrodynamic Separation and Single Molecule Detection.溶液中 DNA-生物分子相互作用的水力分离和单分子检测的多功能分析。
Anal Chem. 2019 Feb 19;91(4):2822-2830. doi: 10.1021/acs.analchem.8b04733. Epub 2019 Feb 6.
2
Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities.用于转录因子结合亲和力定量高通量测量的集成微流控方法
Nucleic Acids Res. 2016 Apr 7;44(6):e51. doi: 10.1093/nar/gkv1327. Epub 2015 Dec 3.
3
Analysis of protein interactions with picomolar binding affinity by fluorescence-detected sedimentation velocity.通过荧光检测沉降速度分析具有皮摩尔结合亲和力的蛋白质相互作用。
Anal Chem. 2014 Mar 18;86(6):3181-7. doi: 10.1021/ac500093m. Epub 2014 Mar 5.
4
DNA-protein interactions: methods for detection and analysis.DNA-蛋白质相互作用：检测和分析方法。
Mol Cell Biochem. 2012 Jun;365(1-2):279-99. doi: 10.1007/s11010-012-1269-z. Epub 2012 Mar 8.
5
Electrokinetic trapping at the one nanometer limit.在纳米极限下的电动捕获。
Proc Natl Acad Sci U S A. 2011 May 31;108(22):8937-42. doi: 10.1073/pnas.1103554108. Epub 2011 May 11.
6
Minimalist proteins: Design of new molecular recognition scaffolds.极简蛋白质：新型分子识别支架的设计
Pure Appl Chem. 2004;76(7-8):1579-1590. doi: 10.1351/pac200476071579.
7
A comparative study of DNA complexation with Mg(II) and Ca(II) in aqueous solution: major and minor grooves bindings.水溶液中DNA与Mg(II)和Ca(II)络合的比较研究：大沟和小沟结合
Biophys J. 2003 Apr;84(4):2460-6. doi: 10.1016/S0006-3495(03)75050-4.
8
The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties.不同MBD蛋白对特定甲基化位点的亲和力取决于它们的内在结合特性。
Nucleic Acids Res. 2003 Mar 15;31(6):1765-74. doi: 10.1093/nar/gkg249.
9
Interaction of RNA with phage display selected peptides analyzed by capillary electrophoresis mobility shift assay.通过毛细管电泳迁移率变动分析来研究RNA与噬菌体展示筛选肽之间的相互作用。
RNA. 2002 May;8(5):698-704. doi: 10.1017/s1355838202020319.
10
Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis.通过傅里叶变换红外光谱和毛细管电泳研究的银（I）与DNA和RNA的配合物。
Biophys J. 2001 Sep;81(3):1580-7. doi: 10.1016/S0006-3495(01)75812-2.
本文引用的文献
1
Coupled folding and site-specific binding of the GCN4-bZIP transcription factor to the AP-1 and ATF/CREB DNA sites studied by microcalorimetry.通过微量热法研究GCN4-bZIP转录因子与AP-1和ATF/CREB DNA位点的耦合折叠和位点特异性结合。
Biochemistry. 1996 Nov 26;35(47):14984-91. doi: 10.1021/bi961312a.
2
Design and optimization of a capillary electrophoretic mobility shift assay involving trp repressor-DNA complexes.涉及色氨酸阻遏物 - DNA 复合物的毛细管电泳迁移率变动分析的设计与优化。
J Chromatogr B Biomed Appl. 1996 Aug 9;683(1):77-84. doi: 10.1016/0378-4347(96)00034-5.
3
DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.
Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):86-90. doi: 10.1073/pnas.93.1.86.
4
Towards more measurement in biology.
Nature. 1994 Mar 10;368(6467):95. doi: 10.1038/368095a0.
5
Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis.分子隔离在聚丙烯酰胺凝胶电泳过程中稳定了CAP-DNA复合物。
Nucleic Acids Res. 1994 Nov 25;22(23):5054-9. doi: 10.1093/nar/22.23.5054.
6
Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis.通过聚丙烯酰胺凝胶电泳研究乳糖阻遏物-操纵基因相互作用的平衡与动力学
Nucleic Acids Res. 1981 Dec 11;9(23):6505-25. doi: 10.1093/nar/9.23.6505.
7
A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.一种用于定量蛋白质与特定DNA区域结合的凝胶电泳方法：应用于大肠杆菌乳糖操纵子调控系统的组分
Nucleic Acids Res. 1981 Jul 10;9(13):3047-60. doi: 10.1093/nar/9.13.3047.
8
Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA.GCN4的DNA结合结构域在与DNA特异性结合时的折叠转变。
Nature. 1990 Oct 11;347(6293):575-8. doi: 10.1038/347575a0.
9
Use of gel retardation to analyze protein-nucleic acid interactions.使用凝胶阻滞分析法分析蛋白质-核酸相互作用。
Microbiol Rev. 1992 Dec;56(4):509-28. doi: 10.1128/mr.56.4.509-528.1992.
10
The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex.GCN4碱性区域亮氨酸拉链以不间断的α螺旋二聚体形式结合DNA：蛋白质-DNA复合物的晶体结构。
Cell. 1992 Dec 24;71(7):1223-37. doi: 10.1016/s0092-8674(05)80070-4.
Zotero 插件