Autor: |
Sirbu BM; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA., Couch FB, Cortez D |
Jazyk: |
angličtina |
Zdroj: |
Nature protocols [Nat Protoc] 2012 Mar 01; Vol. 7 (3), pp. 594-605. Date of Electronic Publication: 2012 Mar 01. |
DOI: |
10.1038/nprot.2012.010 |
Abstrakt: |
Understanding the processes of DNA replication, chromatin assembly and maturation, and the replication stress response requires the ability to monitor protein dynamics at active and damaged replication forks. Detecting protein accumulation at replication forks or damaged sites has primarily relied on immunofluorescence imaging, which is limited in resolution and antibody sensitivity. Here we describe a procedure to isolate proteins on nascent DNA (iPOND) that permits a high-resolution spatiotemporal analysis of proteins at replication forks or on chromatin following DNA replication in cultured cells. iPOND relies on labeling of nascent DNA with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). Biotin conjugation to EdU-labeled DNA using click chemistry facilitates a single-step streptavidin purification of proteins bound to the nascent DNA. iPOND permits an interrogation of any cellular process linked to DNA synthesis using a 3- to 4-d protocol. |
Databáze: |
MEDLINE |
Externí odkaz: |
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