Anti-Ub beads were prepared by coupling 2 mg of anti-Ub Ab to 1 mL of protein G-agarose beads (Roche) with dimethyl pimelimidate according to manufacturer’s instructions (Pierce, Rockford, IL). 0.8 x 4 cm Poly-Prep chromatography columns (Bio-Rad) were then filled with 0.5 mL of anti-Ub beads. Prior to each purification, cell lysates were pre-cleared with columns containing only protein G agarose beads for 10 min. at 4 °C. Pre-cleared lysates containing 12.5 mg of total protein were then loaded onto immunoaffinity purification columns and allowed to incubate for 2 h at 4 °C. Columns were washed 10x with 5 mL of modified RIPA buffer. Bound material was eluted with 2% SDS for 20 min at 37 °C. Eluates from three successive purifications were pooled together and concentrated with an Amicon Ultra-4 10 000 MWCO centrifugal filter (Millipore, Nepean, ON). 5x sample buffer (2% SDS, 10% glycerol, 40 mM Tris pH 6.8, 715 mM β-mercaptoethanol when diluted to 1x) was added to the concentrated samples and proteins were separated on a 10% SDS-PAGE gel and visualized with a Colloidal Coomassie (Invitrogen).
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