Tube2 agarose

At least 700 μg of total protein from mouse primary CD4⁺ T cells was enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit (Cat#662200, Calbiochem, Millipore-Sigma, MO, USA) or TUBE2 Agarose (Cat#UM402, LifeSensors, PA, USA), following the manufacturer’s protocol.

Rat cardiac H9c2 cells were exposed to 10μM MG-132 (a proteasome inhibitor) for 36 h to drastically increase the levels of ubiquinated proteins. Samples were then sonicated in 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol, and protease inhibitors (P2714, Sigma) at 4°C. After centrifugation (14,000xg) for 10 min at 4°C, the supernatant was incubated with Tandem Ubiquitin Binding Entities (TUBEs) bound to agarose (TUBE2-agarose) (UM402, obtained from LifeSensors, PA, USA). TUBEs display a significantly higher increase in affinity for polyubiquitin moieties (up to 1000-fold) over the single ubiquitin binding associated domain (UBA) [21 (link)]. For every mg of total protein, 25 μl of resin was utilized and incubated for 1 h at 4°C with rocking. TUBE-agarose was collected by low speed centrifugation (1000xg, 4°C) for 2 min. The beads were washed with Tris-buffered saline containing 0.05% Tween-20 (TBST) and collected by low speed centrifugation. Washing was repeated three times and the polyubiquinated proteins eluted with 0.2 M glycine HCl, pH 2.5 (3X total volume of pelleted resin) for one 1 h at 4°C. Beads were removed by centrifugation at 10,000xg for 5 min. The eluted polyubiquitinated proteins were quantified, mixed with Laemmli sample buffer and heated at 95°C for 5 min.