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. Author manuscript; available in PMC: 2013 Jun 4.
Published in final edited form as: Nat Protoc. 2012 Mar 1;7(3):594–605. doi: 10.1038/nprot.2012.010

Fig. 4. Example of results obtainable with iPOND.

Fig. 4

Cells were pulsed with EdU for 10 minutes and then incubated with thymidine for 0, 10, or 30 minutes as indicated.. iPOND was performed as described in the protocol. Eluted proteins (lanes 5-8) were analyzed by SDS-PAGE followed by immunoblotting for the replication proteins PCNA, chromatin assembly factor 1 (CAF-1/p60), and histone H2B. As expected, proteins are detectable in every sample of the input (lanes 1-4). In the absence of click chemistry (No Clk, lane 5, negative control), no proteins are isolated from nascent DNA. PCNA and CAF-1 are enriched specifically at the replication fork (Click rxn, lane 6), but not on nascent DNA that is thymidine chased away from the replication fork (Click rxn, lanes 7,8). In contrast, a chromatin bound protein such as H2B is detectable both at the replication fork (Click rxn, lane 6) and in thymidine chased samples (Click rxn, lanes 7,8).