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. 2012 Oct 3;7(10):e46398. doi: 10.1371/journal.pone.0046398

Figure 4. MultiDsks can be used to purify a specific ubiquitylated protein. A.

Figure 4

A schematic of purification of ubiquitylated RNAPII from 4-NQO (10 µg/ml) treated Rpb3-FLAG tagged cells. Cells were grown to logarithmic phase and treated with 4-NQO for one hour before extract was created as described earlier. B Western blot of two-step purification, probed with anti-Rpb1 antibody (4H8) and anti-ubiquitin antibody (P4D1). Lanes correspond to; 1- Input extract, 2- Flow through from MultiDsk resin, 3-MultiDsk resin and bound ubiquitin proteins prior to elution, 4- Gluathione beads post elution, 5- Eluted MultiDsk-ubiquitin conjugates, 6- FLAG Immunoprecipitate and 7- Flow through form the FLAG immunoprecipitate. C Silver stain comparing FLAG immunoprecipitate post MultiDsk enrichment (lane 6 from B) to pure Rpb3-FLAG tagged RNAPII. Different concentrations of pure RNAPII are loaded for comparison. Band at corresponding heights are labelled Rpb1 mono-ubiquitin and Rpb1 poly-ubiquitin.