| Chapter title |
Immunodepletion and Immunopurification as Approaches for CSN Research.
|
|---|---|
| Chapter number | 4 |
| Book title |
Proteostasis
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-3756-1_4 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-3754-7, 978-1-4939-3756-1
|
| Authors |
Amnon Golan, Ning Wei, Elah Pick |
| Editors |
Rune Matthiesen |
| Abstract |
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. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 4 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Professor > Associate Professor | 1 | 25% |
| Researcher | 1 | 25% |
| Student > Doctoral Student | 1 | 25% |
| Unknown | 1 | 25% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 2 | 50% |
| Unknown | 2 | 50% |