In vitro ubiquitination of recombinant Sic1

Polyubiquitinated Sic1 with PY motifs (Ub-Sic1^PY) was prepared as previously described²² with some modifications. Briefly, the Ub conjugation mixture contained 10 pmol Sic1^PY, 2 pmol Uba1, 5 pmol Ubc4, 5 pmol Rsp5 and 1.2 nmol ubiquitin in a buffer of 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM DTT, 5 mM ATP and 10 mM MgCl₂. Conjugation proceeded for 4 h at 25 °C. To purify the conjugates, they were absorbed to a Ni-NTA resin (Qiagen, Hilden, Germany), washed with buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 40% glycerol), eluted with 200 mM imidazole in wash buffer and dialyzed into wash buffer containing 10% glycerol.