Purification of 6His-Ubiquitinated Proteins
Corresponding Organization :
Other organizations : Princeton University
Variable analysis
- Plasmid (YEp352-6HisUb) harboring 6His-tagged ubiquitin under the CUP1-inducible promoter
- Purification of 6His-ubiquitinated proteins
- Western blot analysis using anti-Myc and anti-His antibodies
- Growth media lacking uracil (to select for the plasmid)
- 24 h treatment with 0.1 mM CuSO4 to induce 6His-Ubiquitin
- 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)
- Ni-NTA (Ni2+-nitrilotriacetic acid) agarose beads for purification
- 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) for bead washing
- Protein sample buffer for elution
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