Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

被引：254

作者：

Warren, CM ^{[1
]}

Krzesinski, PR ^{[1
]}

Greaser, ML ^{[1
]}

机构：

[1] Univ Wisconsin, Muscle Biol Lab, Madison, WI 53706 USA

来源：

ELECTROPHORESIS | 2003年 / 24卷 / 11期

关键词：

agarose gel electrophoresis; electroblotting; isoforms; titin;

D O I：

10.1002/elps.200305392

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The electrophoretic separation of high-molecular-weight proteins (>500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SIDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SIDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a similar to13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain (similar to220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.

引用

页码：1695 / 1702

页数：8

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