Bio-Rad Nuvia™ IMAC Resin - Manual (Page 24)

20 Nuvia IMAC Ni-Charged Resin
Materials for On-Column Renaturation
1. Reagents
Refolding buffer
• 20 mM sodium phosphate (NaH
2
PO
4
) (pH 8.0)
• 20 mM imidazole
• 300 mM NaCl
• 1 mM
β-mercaptoethanol
Urea binding buffer (refolding buffer with 8 M urea)
Phosphate elution buffer (with high imidazole)
• 20 mM sodium phosphate (NaH
2
PO
4
) (pH 8.0)
• 500 mM imidazole
• 300 mM NaCl
• 1 mM
β-mercaptoethanol
Note: The β-mercaptoethanol should be added to solutions
only immediately before use.
Method for On-Column Renaturation
1. Wash the column containing the bound protein with 10 column
volumes of urea binding buffer.
2. Apply a linear gradient from 100% urea-binding buffer to 100%
refolding buffer over 60 min at 0.5 ml/min. Refolding is initiated
by a descending gradient from 8 to 0 M urea.
3. Apply a linear gradient from 100% refolding buffer to 100%
phosphate elution buffer with high imidazole.
4. If necessary, add another chromatography step.
Size exclusion chromatography may be a good choice
because aggregates of unrefolded protein can be removed
and the buffer composition of the purified material can be
changed simultaneously.