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Covalent immobilization of antibodies to protein G beads is a basic molecular biology method, although the beads present poor recovery results. Our aim was to reuse the immobilized antibody-protein G complex on a very small scale, therefore we optimized the crosslinking procedure to be used on the wells of a standard 96-well microplate. The method used involves the affinity binding of the antibody to the protein G surface, followed by the immobilization step using different crosslinking reagents, DMP and BS3, quenching the crosslinking reaction, and binding the antibody-specific antigen. By scaling down the procedure, we were able to reuse the anti-EGFR crosslinked wells more than 20 times. This method can be used to perform assays on a wide range of solid supports containing the protein G in an immobilized form, including functionalized nanosensors, for immunoprecipitation, protein and cell lysate purification, target protein enrichment. Copyright © 2020 Elsevier B.V. All rights reserved.


Mónika Korodi, Kinga Rákosi, Mihaela Baibarac, Szilard N Fejer. Reusable on-plate immunoprecipitation method with covalently immobilized antibodies on a protein G covered microtiter plate. Journal of immunological methods. 2020 Aug;483:112812

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PMID: 32569597

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