A novel strategy for quantitative isoform detection directly performed from culture supernatant.
Currently, one of the most used techniques for the determination of isoform pattern analysis is isoelectric focusing. Routinely, this is performed by immunoblotting. Blotting of proteins after isoelectric focusing on IPG gels may cause several problems, such as protein loss by the blotting itself and band broadening, in some cases the immunostaining with antibodies might be problematic. In the present study, an alternative isoform prestaining method with CyDye fluors is presented. For this approach, a highly glycosylated fusion protein, Epo-Fc, was used consisting of two recombinant human erythropoietin attached to the Fc part of a human IgG(1) molecule. By using CyDye fluors, up to three samples can be focused on the same lane under identical electrophoretic conditions. A fundamental benefit of this technique is the ability to perform quantitative isoform pattern analysis directly from serum-free culture supernatant.
Schriebl
Kornelia
K
Austrian Center of Biopharmaceutical Technology, Muthgasse 18, A-1190 Vienna, Austria. kschriebl@gmx.at
Trummer
Evelyn
E
Weik
Robert
R
Müller
Dethardt
D
Kunert
Renate
R
Lattenmayer
Christine
C
Katinger
Hermann
H
Vorauer-Uhl
Karola
K
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004921:Erythropoietin / Q000032:analysis*; D007141:Immunoglobulin Fc Fragments / Q000032:analysis*; D007525:Isoelectric Focusing; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D011994:Recombinant Proteins; D015203:Reproducibility of Results
D007141:Immunoglobulin Fc Fragments; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004921:Erythropoietin