FLAG-tagged 26S proteasomes were incubated with Rad23 and fluorescently labeled Ub(n)-TAIL or Ub(n)-FOLD in the presence of ADP. BeFx (1st incub.). Proteasomes were retrieved with beads containing FLAG antibodies, washed, and incubated with an excess of free ubiquitin chains (Ub(n)) for different time periods (2nd incub.). The bead-bound material was analyzed by SDS-PAGE, followed by fluorescence scanning and Coomassie-blue staining. The bound substrate was quantified relative to the material bound in the absence of Ub(n) (lanes 1 and 6 set to 100%).
