MultiDsks can be used to characterise the kinetics of ubiquitylation of a specific protein species. A.

<p>Exponentially growing yeast cells were either treated with 10 µg/ml of 4-NQO for one hour, or left untreated, as indicated. Equal amounts of extracts were incubated with MultiDsk resin. Dilute Input extract (1%) and washes from the beads were also loaded. Proteins were analysed by Western blotting using anti-Rpb1 antibody, 4H8. <b>B</b>. Exponentially growing yeast cells were either treated with 10 µg/ml of 4-NQO for one hour, or left untreated, as indicated. Equal amounts of the extracts were incubated with agarose beads loaded with GST alone, GST-Dsk2 protein, commercial TUBE1, or MultiDsk. Proteins were eluted via boiling in sample buffer and subjected to Western blot analysis using the anti-Rpb1 antibody, 4H8 (upper panel). Ponceau S staining (lower panel) shows relative amounts of affinity proteins used. <b>C</b>. Yeast cells were harvested at the indicated time after treatment with 4-NQO (10 µg/ml), incubated with MultiDsk resin, and isolated proteins were analysed by Western blotting using either 4H8, or an anti-Def1 antibody, as indicated.</p

MultiDsk binds efficiently to ubiquitylated proteins. A

<p>. Schematic representation of the MultiDsk protein. <b>B</b>. 2 mg of yeast whole cell lysate from strain SUB592 expressing Myc-His-tagged ubiquitin was incubated with affinity beads. Differing amounts of GST protein, alone or as a mixture with GST-MultiDsk protein, were mixed with agarose beads so that equal amounts of total protein and bead bed volumes were used in each experiment. The flow-through not bound to the beads was retained and loaded at equivalent levels to the Input (Lane 1). After Western transfer, the membrane was stained with Ponceau S to reveal total protein in samples (lower panel), and Western blot was performed using anti-Myc antibodies to detect ubiquitylated species (upper panel). <b>C</b>. As in B, except performed on a human cell whole cell extract. 100 µg of protein was incubated with the indicated amounts of GST, GST-Dsk2, or MultiDsk protein and purified via agarose beads. <b>D</b>. As in B, but using purified ubiquitin chains. GST alone, full length GST-Dsk2 protein, or MultiDsk, bound to beads, were incubated with 100 µg synthetic K48- or K63-linked ubiquitin chains for 2 hours. After Western transfer, the membrane was stained with Ponceau S to reveal total protein in samples (lower panel), and Western blot was performed using anti-ubiquitin antibodies to detect ubiquitylated species (upper panel).</p

Protection of poly-ubiquitin chains in extract.

<p>Extract from strain SUB592 was incubated with equivalent amounts of GST, GST-Dsk2, commercially available TUBE-1, and MultiDsk and incubated at 30°C for the indicated time. Total protein extracts were subject to Western blot and probed using anti-myc antibody.</p