Purification of 6His-Ubiquitinated Proteins

Cells were transformed with a plasmid (YEp352-6HisUb) harbouring 6His-tagged ubiquitin under the CUP1-inducible promoter51 (link). The cells were grown in media lacking uracil (to select for the plasmid). 6His-Ubiquitin was induced by 24 h treatment with 0.1 mM CuSO₄. Cells were harvested and lysed with guanidinium lysis buffer (6 M guanidine hydrochloride, 100 mM sodium phosphate buffer pH8.0, 10 mM Tris-HCl pH8.0, 10 mM imidazole, 10 mM β-mercaptoethanol, 0.1% Triton X-100, 2.5 mg ml⁻¹ N-methyl maleimide, 0.1 mM MG-132, 1 × protease inhibitor). Purification of 6His-ubiquitinated proteins was performed using the Ni-NTA (Ni²⁺-nitrilotriacetic acid) agarose beads (QIAGEN). The beads were washed with urea buffer (8 M urea, 100 mM sodium phosphate buffer pH 6.4, Tris-HCl pH 6.4, 10 mM imidazole, 10 mM β-mercaptoethanol, 0.1% Triton X-100) and subsequently eluted with protein sample buffer. Eluted protein samples were separated by SDS–PAGE and analysed by western blots using anti-Myc (Clontech) and anti-His antibodies (Novagen).