Zitieren Sie bitte immer diesen URN: urn:nbn:de:kobv:b43-479786
Preactivation Crosslinking - An Efficient Method for the Oriented Immobilization of Antibodies
- Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and orientedCrosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.…
Autor*innen: | Barbara Schroeder, Hoa Le Xuan, Jule L. Völzke, Michael G. WellerORCiD |
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Dokumenttyp: | Zeitschriftenartikel |
Veröffentlichungsform: | Verlagsliteratur |
Sprache: | Englisch |
Titel des übergeordneten Werkes (Englisch): | Methods and Protocols |
Jahr der Erstveröffentlichung: | 2019 |
Organisationseinheit der BAM: | 1 Analytische Chemie; Referenzmaterialien |
1 Analytische Chemie; Referenzmaterialien / 1.5 Proteinanalytik | |
Veröffentlichende Institution: | Bundesanstalt für Materialforschung und -prüfung (BAM) |
Verlag: | MDPI |
Verlagsort: | Basel, Switzerland |
Jahrgang/Band: | 2 |
Ausgabe/Heft: | 2 |
Erste Seite: | 1 |
Letzte Seite: | 14 |
DDC-Klassifikation: | Naturwissenschaften und Mathematik / Chemie / Analytische Chemie |
Technik, Medizin, angewandte Wissenschaften / Ingenieurwissenschaften / Ingenieurwissenschaften und zugeordnete Tätigkeiten | |
Freie Schlagwörter: | Affinity chromatography; Antibody coating; Bioconjugation; Biosensor; IgG; Immunoaffinity extraction; Immunoassay; Immunocapture; Immunoglobulins; Immunoprecipitation; Immunosensor; Oriented immobilization; Protein A; Protein G; Proximity-enhanced reaction; Regeneration |
Themenfelder/Aktivitätsfelder der BAM: | Chemie und Prozesstechnik |
Material | |
Material / Nano | |
DOI: | 10.3390/mps2020035 |
URN: | urn:nbn:de:kobv:b43-479786 |
ISSN: | 2409-9279 |
Zugehöriger Identifikator: | https://nbn-resolving.org/urn:nbn:de:kobv:b43-478797 |
Verfügbarkeit des Dokuments: | Datei für die Öffentlichkeit verfügbar ("Open Access") |
Lizenz (Deutsch): | Creative Commons - CC BY - Namensnennung 4.0 International |
Datum der Freischaltung: | 20.05.2019 |
Referierte Publikation: | Nein |
Schriftenreihen ohne Nummerierung: | Wissenschaftliche Artikel der BAM |