title: Applicability of different fluorescent dyes for isoform quantification on linear IPG gels.
abstract: For biotechnological research, development, and production various analytical methods are required to determine the quality of the target product. In this context, the determination of isoforms is state-of-the-art; however, the majority of applied techniques are more qualitative than quantitative. To address this fact, we evaluated different post- and pre-electrophoretic staining dyes for their applicability on linear IPG gels using recombinant human erythropoietin as a model protein. Each evaluated dyes was able to detect all isoforms reproducibly, but CyDyes were found to be the most promising. Using CyDyes, up to three samples can be focused on the same lane under identical electrophoretic conditions, thus, a fast, reproducible, sensitive and quantitative isoform determination can be performed. To illustrate the practical relevance, quantitative CyDye technique was used for the characterization of our model protein, recombinant human Epo-Fc. This method makes it possible to determine the isoform pattern of nonpurified supernatants as well as purified proteins. We conclude that quantitative pre-electrophoretic staining IEF using CyDyes is a fast, simple, accurate method to determine isoforms, which can be used in research, development, and manufacturing for product quality analysis, e.g., clone screening, process optimization, and purification monitoring.
authors.affiliation: Austrian Center of Biopharmaceutical Technology, 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: Christine
authors.initials: C
authors.lastname: Lattenmayer
authors.forename: Dethardt
authors.initials: D
authors.lastname: Müller
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
authors.forename: Hermann
authors.initials: H
authors.lastname: Katinger
authors.forename: Karola
authors.initials: K
authors.lastname: Vorauer-Uhl
mesh_terms: D002232:Carbocyanines / Q000737:chemistry*; D003720:Densitometry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis / Q000737:chemistry; D005063:Europium / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D020033:Protein Isoforms; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D020033:Protein Isoforms; D011506:Proteins; D011994:Recombinant Proteins; C089187:cyanine dye 3; C085321:cyanine dye 5; D004921:Erythropoietin; D005063:Europium