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Das Ziel dieser Arbeit war es, die Vernetzung (Crosslinking) zwischen Protein G und einem Antikörper so durchzuführen, dass es zu keiner Modifikation der Antigen-Bindungsstelle des Antikörpers kommt. Zudem wurde im Rahmen dieser Arbeit ein Immunoassay entwickelt, mithilfe dessen zwanzig verschiedene Crosslinker in Hinblick auf ihre Fähigkeit Protein G zu funktionalisieren, untersucht werden konnten. Es konnten fünf Reagenzien identifiziert werden, die eine Derivatisierung von Protein G sowie eine anschließende Immobilisierung des Antikörpers auf dem Protein ermöglichen. Des Weiteren wurde gezeigt, dass mithilfe dieses Verfahrens sowohl die Immunpräzipitation als auch die massenspektrometrische Identifizierung und Charakterisierung von Proteinkomplexen erleichtert und verbessert werden kann.
Fast and robust purification of antibodies from human serum with a new monolithic protein A column
(2017)
The use of antibodies is becoming more and more important. Therewith the need for fast and robust methods for their purification and columns with a long lifetime is growing. In this application, we describe a fast and robust protein A affinity chromatography method with a new monolithic column. Antibodies were successfully purified in less than 1.5 minutes with a flowrate of 5 mL/min highlighting the advantages of this column and method.
The rational design of bright optical probes and dye–biomolecule conjugates in the NIR-region requires fluorescent labels that retain their high fluorescence quantum yields when bound to a recognition unit or upon interaction with a target. Because hydrophilicity-controlled dye aggregation in conjunction with homo-FRET presents one of the major fluorescence deactivation pathways in dye–protein conjugates, fluorescent labels are required that enable higher labeling degrees with minimum dye aggregation. Aiming at a better understanding of the factors governing dye–dye interactions, we systematically studied the signal-relevant spectroscopic properties, hydrophilicity, and aggregation behavior of the novel xS-IDCC series of symmetric pentamethines equipped with two, four, and six sulfonic acid groups and selected conjugates of these dyes with IgG and the antibody cetuximab (ctx) directed against the cancer-related epidermal growth factor (EGF) receptor in comparison to the gold standard Cy5.5. With 6S-IDCC, which displays a molar absorption coefficient of 190 000 M1 cm1 and a fluorescence quantum yield (Φf) of 0.18 in aqueous media like PBS and nearly no aggregation, we could identify a fluorophore with a similarly good performance as Cy5.5. Bioconjugation of 6S-IDCC and Cy5.5 yielded highly emissive targeted probes with comparable Φf values of 0.29 for a dye-to-protein (D/P) ratio <1 and a reduced number of protein-bound dye aggregates in the case of 6S-IDCC. Binding studies of the ctx conjugates of both dyes performed by fluorescence microscopy and FACS revealed that the binding strength between the targeted probes and the EGF receptor at the cell membrane is independent of D/P ratio. These results underline the importance of an application-specific tuning of dye hydrophilicity for the design of bright fluorescent reporters and efficient optical probes. Moreover, we could demonstrate the potential of fluorescence spectroscopy to predict the size of fluorescence signals resulting for other fluorescence techniques such as FACS.