Improved Antibody Immobilization Using Aniline-Catalyzed Aldehyde-Hydrazide Chemistry For The Study Of Protein-Protein Interactions

Covalent attachment of an antibody to a beaded support before immunoprecipitation prevents antibody contamination upon antigen elution and allows for the detection of antigens with similar molecular weights as antibody heavy and light chains. Common methods of antibody immobilization include direct coupling of antibody amino groups to aldehyde supports followed by reduction and indirect coupling of antibodies to Protein A/G‐coated beads followed by amine‐reactive crosslinking. Drawbacks of these methods include the loss of antibody functionality due to chemical modification or obstruction of the antigen binding site. An alternative approach for the covalent attachment of antibody to a beaded support utilizes oxidization of sugars in the antibody Fc region to aldehydes, which react with a hydrazide‐activated support. This method allows for unobstructed and oriented antibody conjugation without adverse modifications to antigen binding sites. However, compared to amine‐reactive antibody crosslinking, aldehyde‐hydrazide coupling often has lower efficiency and requires significantly longer incubation times. In order to improve this conjugation chemistry, we developed new aldehyde‐hydrazide immobilization and immunoprecipitation kits. These kits include aniline as a catalyst in a buffer with optimized pH for increased antibody coupling efficiency. The addition of aniline results in an increase of coupling efficiency from 50% to 90% for most antibody subtypes, and shortens reaction times to less than 4 hours. In addition, we demonstrate improved immunoprecipitation of Epitheleal Growth Factor Receptor (EGFR) and Signal Transducers and Activator of Transcription 1 Protein (STAT1), as well as the co‐immunoprecipitation of endogenous Cyclin B1/Cyclin Dependant Kinase 1 (Cdk1) complex from cell lysates using 2–10μg of immobilized antibody.