IgM characterization directly performed in crude culture supernatants by a new simple electrophoretic method.
A new electrophoretic technique for the qualitative and quantitative analyses of IgM isoforms and fragments has been developed. IgMs which are more complex than many other recombinantly expressed immunoglobulins are characterized by their high molecular weighted active forms and many additional isoforms and fragments in the molecular range between 25 and 1200kDa. To analyze the multimers, isoforms and fragments simultaneously a high-resolution method, which enables sufficient migration and separation is required. Furthermore, this method should be appropriate to analyze IgMs in crude culture supernatants as well as purified samples. Simple sample preparation avoiding unspecific protein loss has been established. Currently no standard method to analyze all of them accordingly is available. The IgM-SDS-PAGE investigated for this purpose includes all these aspects. The combination of simple sample preparation and the application of precast gels make this electrophoretic method suitable for research but also quality control. The selective quantification of the multimers and the relative isoform distribution were performed by sensitive Sypro Ruby staining obtaining reliable and reproducible data in clone screening and process development which has been demonstrated by recombinantly expressed IgMs with significantly different isoform pattern.
Vorauer-Uhl
Karola
K
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. Karola.Vorauer-Uhl@boku.ac.at
Wallner
Jakob
J
Lhota
Gabriele
G
Katinger
Hermann
H
Kunert
Renate
R
D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques; D002465:Cell Movement; D002469:Cell Separation; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005782:Gels / Q000737:chemistry; D007075:Immunoglobulin M / Q000032:analysis* / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology; D012680:Sensitivity and Specificity
D017077:Culture Media, Conditioned; D005782:Gels; D007075:Immunoglobulin M; D010446:Peptide Fragments; D020033:Protein Isoforms