Isolation of isoproteins from monoclonal antibodies and recombinant proteins by chromatofocusing.
A fast protein liquid chromatographic method for the preparative separation of the various isoproteins is described. Highly purified human monoclonal antibodies, recombinant human superoxide dismutase and human superoxide dismutase from erythrocytes were used as starting material. The isoproteins were separated by chromatofocusing on Mono P columns. A very narrow pH gradient was applied to achieve complete separation of the isoproteins. The prepurification steps and the pretreatment of the samples to achieve optimum resolution are described in detail. The method is also applicable to extremely basic monoclonal antibodies (pI = 9). The successful separation was checked by isoelectric focusing in immobilized pH gradients (Immobilines). The future of these methods is discussed, because for many different biochemical and biophysical investigations pure and homogeneous isoproteins are necessary.
Jungbauer
A
A
Institute of Applied Microbiology, University of Agriculture and Forestry and Biotechnology, Vienna, Austria.
Tauer
C
C
Wenisch
E
E
Uhl
K
K
Brunner
J
J
Purtscher
M
M
Steindl
F
F
Buchacher
A
A
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D011506:Proteins / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013482:Superoxide Dismutase / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D011506:Proteins; D011994:Recombinant Proteins; D013482:Superoxide Dismutase