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In Vivo Protein Cross-Linking and Coimmunoprecipitation in Haloferax volcanii

Authors :
Roberto A, Paggi
Rosana E, De Castro
Micaela, Cerletti
Source :
Methods in molecular biology (Clifton, N.J.). 2522
Publication Year :
2022

Abstract

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.

Details

ISSN :
19406029
Volume :
2522
Database :
OpenAIRE
Journal :
Methods in molecular biology (Clifton, N.J.)
Accession number :
edsair.pmid..........ed5dd01677fc66ee1bda42c55ef70cd8