Resolution of DNA topoisomerase II by two-dimensional polyacrylamide gel electrophoresis and western blotting.
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.
Wright
W D
WD
Washington University School of Medicine, Mallinckrodt Institute of Radiology, St. Louis, Missouri 63108.
Roti Roti
J L
JL
D015153:Blotting, Western / Q000379:methods*; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005069:Evaluation Studies as Topic; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D012995:Solubility
D004250:DNA Topoisomerases, Type II