| Chapter title |
Immunoprecipitation of Cullin-RING Ligases (CRLs) in Arabidopsis thaliana Seedlings
|
|---|---|
| Chapter number | 2 |
| Book title |
Plant Proteostasis
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-3759-2_2 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-3757-8, 978-1-4939-3759-2
|
| Authors |
Anna Franciosini, Giovanna Serino |
| Editors |
L. Maria Lois, Rune Matthiesen |
| Abstract |
CRL (Cullin-RING ubiquitin ligase) is the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-RING Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 8 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 2 | 25% |
| Student > Bachelor | 2 | 25% |
| Other | 1 | 13% |
| Student > Master | 1 | 13% |
| Unknown | 2 | 25% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 4 | 50% |
| Chemical Engineering | 1 | 13% |
| Agricultural and Biological Sciences | 1 | 13% |
| Unknown | 2 | 25% |