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Abstract: Background. Protein complexes provide valuable biological information but can be difficult to handle [1]. Therefore, technical advancements designed to improve their manipulation are al-ways useful. Methods. We investigated the opportunity to exploit native agarose gels and the contact bot method for the transfer of native proteins to membranes as means for optimizing the conditions for obtaining stable complexes [2]. As a simple model of protein-protein interactions, an anti-gen-ligand complex was used in which both proteins were fused to reporters. Results. At each step, it was possible to visualize both the antigen, fused to a fluorescent pro-tein, and the ligand, fused to a monomeric ascorbate peroxidase (APEX) and, as such, a way to tune the protocol. The conditions for the complex formation were adapted by modifying the buffer conditions, the concentration of the proteins and of the cross-linkers. Conclusions. The procedure is rapid, inexpensive, and the several detection opportunities al-lows for both the monitoring of complex stability and the preservation of the functionality of its components, which is critical for understanding their biomedical implications and supporting drug discovery. The overall protocol represents a handy alternative to gel filtration, uses very standard and ubiquitous equipment, and can be implemented rapidly and without specific train-ing. References: [1] O. Puig, F. Caspary, G. Rigaut, B. Rutz, E. Bouveret, E. Bragado-Nilsson, M. Wilm, B. Séraphin. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods. (San Diego, Calif.), 24(3), 218–229 [2] C. Sakuma, M. Nakagawa, Y. Tomioka, T. Maruyama, K. Entzminger, J.K. Fleming, T. Shibata, Y. Kurosawa, C.J. Okumura, T. Arakawa, T. Akuta. Western blotting of native proteins from agarose gels. Biotechniques. 2022 May;72(5):207-218
Keywords: protein complexes, contact blotting, native agarose gels, protein interaction
Published in RUNG: 17.06.2024; Views: 208; Downloads: 0
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