Fluorescently labeled Ub(n)-TAIL was incubated with 26S proteasomes, Cdc48-UN, and different concentrations of SBP-tagged Ubx5 or SBP-tagged Rad23. The concentrations of the cofactors are given relative to that of 26S proteasomes. After 60 min, the samples were analyzed by SDS-PAGE, followed by fluorescence scanning and blotting for SBP. The percentage of substrate degraded was quantified by determining the fluorescence in peptides (after background subtraction) and comparing it with the total fluorescence in each lane of the SDS gel.
