Reusable on-plate immunoprecipitation method with covalently immobilized antibodies on a protein G covered microtiter plate

• Published in: Journal of immunological methods Vol. 483; p. 112812
• Main Authors: Korodi, Mónika, Rákosi, Kinga, Baibarac, Mihaela, Fejer, Szilard N.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2020
• Subjects: Antibodies, Immobilized - immunology; Antibody; Antibody Affinity; Antibody Specificity; Antigen-Antibody Complex; Bacterial Proteins - immunology; BS3; DMP; Enzyme-Linked Immunosorbent Assay - instrumentation; Equipment Design; Equipment Reuse; ErbB Receptors - analysis; ErbB Receptors - immunology; Humans; Immunoprecipitation - instrumentation; On-plate crosslinking; Protein G; Thyrotropin - analysis; Thyrotropin - immunology; PBS; RT; BS3; Antibody; Protein G; DMP; HRP; TSH; IgG; EGFR; BSA; OD; On-plate crosslinking; ELISA; BS
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2020.112812

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.