使用固定化pH梯度改善二维电泳中蛋白质的溶解性
Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients.
作者信息
Rabilloud T, Adessi C, Giraudel A, Lunardi J
机构信息
CEA-Laboratoire de Bioénergétique Cellulaire et Pathologique, UA 2019 DBMS/BECP, Grenoble, France.
出版信息
Electrophoresis. 1997 Mar-Apr;18(3-4):307-16. doi: 10.1002/elps.1150180303.
Abstract
Membrane and nuclear proteins of poor solubility have been separated by high resolution two-dimensional (2-D) gel electrophoresis. Isoelectric focusing with immobilized pH gradients leads to severe quantitative losses of proteins in the resulting 2-D map, although the resolution is usually high. Protein solubility could be improved by using denaturing solutions containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and detergents (both nonionic and zwitterionic). The usefulness of thiourea-containing denaturing mixtures is shown for microsomal and nuclear proteins as well as for tubulin, a protein highly prone to aggregation.
摘要
溶解度低的膜蛋白和核蛋白已通过高分辨率二维（2-D）凝胶电泳分离。使用固定化pH梯度进行等电聚焦会导致所得二维图谱中的蛋白质出现严重的定量损失，尽管分辨率通常较高。通过使用含有各种去污剂和离液剂的变性溶液可以提高蛋白质的溶解度。含有尿素、硫脲和去污剂（非离子型和两性离子型）的变性溶液取得了最佳效果。含硫脲的变性混合物对微粒体蛋白、核蛋白以及微管蛋白（一种极易聚集的蛋白质）均显示出有效性。
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本文引用的文献
1
The relationship of structure to the effectiveness of denaturing agents for proteins.蛋白质变性剂的结构与其有效性之间的关系。
Biochemistry. 1963 Jan-Feb;2:47-57. doi: 10.1021/bi00901a011.
2
3
Two-dimensional electrophoresis of membrane proteins: a current challenge for immobilized pH gradients.
Electrophoresis. 1997 Jan;18(1):127-35. doi: 10.1002/elps.1150180124.
4
Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two-dimensional gel electrophoresis.
Electrophoresis. 1995 Oct;16(10):1935-45. doi: 10.1002/elps.11501601320.
5
Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins.
Electrophoresis. 1993 Dec;14(12):1375-8. doi: 10.1002/elps.11501401212.
6
Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension.通过胶内复水进行样品上样可提高在第一维使用固定化pH梯度的双向电泳分辨率。
Electrophoresis. 1994 Dec;15(12):1552-8. doi: 10.1002/elps.11501501223.
7
N-D-Gluco-N-methylalkanamide compounds, a new class of non-ionic detergents for membrane biochemistry.N-D-葡萄糖-N-甲基链烷酰胺化合物，一类用于膜生物化学的新型非离子洗涤剂。
Biochem J. 1982 Nov 1;207(2):363-6. doi: 10.1042/bj2070363.
8
Sulfobetaine derivatives of bile acids: nondenaturing surfactants for membrane biochemistry.胆汁酸的磺基甜菜碱衍生物：用于膜生物化学的非变性表面活性剂。
Anal Biochem. 1983 Apr 1;130(1):72-82. doi: 10.1016/0003-2697(83)90651-6.
9
Preparative isoelectric focusing in immobilized pH gradients. II. A case report.
J Biochem Biophys Methods. 1983 Sep;8(2):157-72. doi: 10.1016/0165-022x(83)90041-6.
10
Two-dimensional electrophoresis of membrane proteins. Factors affecting resolution of rat-liver microsomal proteins.膜蛋白的双向电泳。影响大鼠肝脏微粒体蛋白分辨率的因素。
Biochim Biophys Acta. 1983 Oct 12;734(2):160-7. doi: 10.1016/0005-2736(83)90114-1.
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