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Resolution of DNA topoisomerase II by two-dimensional polyacrylamide gel electrophoresis and western blotting.

Authors
Type
Published Article
Journal
Analytical biochemistry
Publication Date
Volume
204
Issue
1
Pages
124–130
Identifiers
PMID: 1325132
Source
Medline
License
Unknown

Abstract

Eukaryotic DNA Topoisomerase II (Topo II) has been studied using high-resolution two-dimensional polyacrylamide electrophoresis (2D-PAGE) and immunodetection of resolved proteins using specific antisera (Western blotting). Traditional methods of 2D-PAGE failed to resolve Topo II and neither nonequilibrium nor equilibrium pH gradients allowed Topo II to enter the first dimension gel. Exhaustive nuclease digestion and alternate protein solubilization strategies also produced negative results. We have developed altered first dimension pH gradient profiles and employed a more aggressive protein solubilization procedure which resulted in the resolution of Topo II. The 170-kDa polypeptide focuses with an apparent isoelectric point of approximately 6.5.

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