title: A novel strategy for quantitative isoform detection directly performed from culture supernatant.
abstract: 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.
authors.affiliation: Austrian Center of Biopharmaceutical Technology, Muthgasse 18, A-1190 Vienna, Austria. kschriebl@gmx.at
authors.forename: Kornelia
authors.initials: K
authors.lastname: Schriebl
authors.forename: Evelyn
authors.initials: E
authors.lastname: Trummer
authors.forename: Robert
authors.initials: R
authors.lastname: Weik
authors.forename: Dethardt
authors.initials: D
authors.lastname: Müller
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
authors.forename: Christine
authors.initials: C
authors.lastname: Lattenmayer
authors.forename: Hermann
authors.initials: H
authors.lastname: Katinger
authors.forename: Karola
authors.initials: K
authors.lastname: Vorauer-Uhl
mesh_terms: 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
chemical_list: D007141:Immunoglobulin Fc Fragments; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004921:Erythropoietin