Immunodepletion and Immunopurification as Approaches for CSN Research.
The COP9 signalosome (CSN) is an evolutionary conserved complex that is found in all eukaryotes, and implicated in regulating the activity of Cullin-RING ubiquitin Ligases (CRLs). Activity of CRLs is highly regulated; complexes are active when the cullin subunit is covalently attached to the ubiquitin like modifier, Nedd8. Neddylation/deneddylation cycles are required for proper CRLs activity, and deneddylation is performed by the CSN complex.We describe here a method utilizing resin-coupled antibodies to deplete the CSN from human cell extracts, and to obtain endogenous CSN complexes by immunopurification. In the first step, the cross-linked primary antibodies recognize endogenous CSN complexes, and deplete them from cell extract as the extract passes through the immunoaffinity column. The resulting "CSN-depleted extract" (CDP) is rich in neddylated cullins that can be used as a substrate for cullin-deneddylation assay for CSN complexes purified from various eukaryotes. Consequently, regeneration of the column results in dissociation of a highly purified CSN complex, together with its associated proteins. Immunopurification of the CSN from various human tissues or experimental conditions is advantageous for the generation of numerous CSN-interaction maps.
Department of Biology and Environment, University of Haifa at Oranim, Tivon, 3600600, Israel.|Protein Purification Center, Ramat Yohanan, 3003500, Israel.
Amnon
A
Golan
Department of Molecular, Cellular and Developmental Biology, Yale University, Prospect St, New Haven, CT, 06511-2106, USA.
Ning
N
Wei
Department of Biology and Environment, University of Haifa at Oranim, Tivon, 3600600, Israel. elahpic@research.haifa.ac.il.
Elah
E
Pick
D000818:Animals; D035843:Biomedical Research / Q000379:methods*; D000075686:COP9 Signalosome Complex / Q000235:genetics / Q000378:metabolism*; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D000075687:NEDD8 Protein / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D044842:Cullin Proteins; D000075687:NEDD8 Protein; D044767:Ubiquitin-Protein Ligases; D000075686:COP9 Signalosome Complex
COP9 signalosome; Cullin-RING ubiquitin ligase; Immunodepletion; Immunopurification; Nedd8