Abstract
DNA replication is a highly complex process that achieves the faithful transmission of genetic information from parent to progeny. Recruitment of DNA replication proteins to DNA is dynamically regulated during the cell cycle and in response to replication stresses. For a large-scale analysis of DNA replication proteins, I established a method for analysis of chromatin-bound proteins by SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomics. Here I describe a detailed methodology for SILAC labeling of budding yeast Saccharomyces cerevisiae, then nuclear isolation and chromatin preparation from synchronized yeast cells, prior to quantitative proteomic analysis of DNA replication proteins.
| Original language | English |
|---|---|
| Title of host publication | SILAC |
| Subtitle of host publication | Methods and Protocols |
| Editors | Jose L. Luque-Garcia |
| Place of Publication | New York, NY |
| Publisher | Springer US |
| Chapter | 17 |
| Pages | 209-218 |
| Number of pages | 10 |
| Edition | 1 |
| ISBN (Electronic) | 978-1-0716-2863-8 |
| ISBN (Print) | 978-1-0716-2862-1 |
| DOIs | |
| Publication status | Published - 13 Nov 2022 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Publisher | Springer |
| Volume | 2603 |
| ISSN (Print) | 1064-3745 |
| ISSN (Electronic) | 1940-6029 |
Bibliographical note
AcknowledgmentsI thank Anne Donaldson (University of Aberdeen) for reading the manuscript. The method described in this chapter was established and funded by Cancer Research UK grants C1445/A8791 and C1445/A11646 and BBSRC grant BB/K006304/1.
Keywords
- SILAC
- quantitative proteomics
- DNA replication
- Saccharomyces cerevisiae
- Chromatin
- nuclear isolation
