About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis

被引：41

作者：

Luche, S

Diemer, H

Tastet, C

Chevallet, M

Van Dorsselaer, A

Leize-Wagner, E

Rabilloud, T

机构：

[1] BECP, DRDC, Lab Bioenerget Cellulaire & Pathol, CEA, F-38054 Grenoble 9, France

[2] ECPM, CNRS, Lab Spectrometrie Masse Bioorgan, UMR 7509, Strasbourg, France

来源：

PROTEOMICS | 2004年 / 4卷 / 03期

关键词：

basic proteins; cysteine; maleimide; two-dimensional gel electrophoresis PRO 0589;

D O I：

10.1002/pmic.200300589

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The influence of thiol blocking on the resolution of basic proteins by two-dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in-gel rehydration on wide gradients (e.g. 3-10 and 4-12), but anodic cup-loading was still required for basic gradients (e.g. 6-12 or 8-12). These results demonstrate that thiol-related problems are not solely responsible for streaking of basic proteins on two-dimensional gels.

引用

页码：551 / 561

页数：11

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