In vivo Protein Crosslinking and Co-Immunoprecipitation in Haloferax volcanii

Abstract

Co-Immunoprecitation is a powerful and commonly used method to identify protein-protein interactions in a physiological context. Here, we report a Co-Immunoprecitation Co-IP 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 crosslinking 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.