In Vivo Protein Cross-Linking and Coimmunoprecipitation in Haloferax volcanii

ROBERTO A. PAGGI; ROSANA E. DE CASTRO; MICAELA CERLETTI

Archaea: Methods and Protocols

Springer Nature

Año: 2022; p. 301 - 317

Coimmunoprecipitation is a powerful and commonly used method to identify protein–protein interactions in a physiological context. Here, we report a coimmunoprecipitation protocol that was adapted and optimized for the haloarchaeon Haloferax volcanii to identify interacting partners to the LonB protease. This protocol includes the in vivo cross-linking of H. volcanii proteins using two different crosslinker agents, dithiobis(succinimidyl propionate) and formaldehyde, followed by immunoprecipitation with anti-LonB antibody conjugated to Protein A − Sepharose beads. Tryptic on-bead protein digestion was performed combined with Mass Spectrometry analysis of peptides for the identification and quantification of LonB ligands. *Cerletti M es la autora correspondiente.