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Preactivation Crosslinking—An Efficient Method for the Oriented Immobilization of Antibodies

1
Federal Institute for Materials Research and Testing (BAM), Division 1.5 Protein Analysis, Richard-Willstätter-Strasse 11, 12489 Berlin, Germany
2
Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Strasse 2+4, 14195 Berlin, Germany
*
Author to whom correspondence should be addressed.
Methods Protoc. 2019, 2(2), 35; https://doi.org/10.3390/mps2020035
Received: 16 April 2019 / Revised: 24 April 2019 / Accepted: 29 April 2019 / Published: 3 May 2019
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 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. View Full-Text
Keywords: antibody coating; proximity-enhanced reaction; immunoglobulins; IgG; Protein A; Protein G; bio-interaction; immunoprecipitation; pull-down assay; immunocapture; stabilization; yield; regeneration; nanoparticles; microparticles; biochips; immunosensor; photochemical crosslinker; click chemistry; Herceptin; Trastuzumab antibody coating; proximity-enhanced reaction; immunoglobulins; IgG; Protein A; Protein G; bio-interaction; immunoprecipitation; pull-down assay; immunocapture; stabilization; yield; regeneration; nanoparticles; microparticles; biochips; immunosensor; photochemical crosslinker; click chemistry; Herceptin; Trastuzumab
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Schroeder, B.; Le Xuan, H.; Völzke, J.L.; Weller, M.G. Preactivation Crosslinking—An Efficient Method for the Oriented Immobilization of Antibodies. Methods Protoc. 2019, 2, 35.

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