Bio-Rad Profinity IMAC Resins - Manual (Page 32)

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• Elution buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– Higher concentration of imidazole* (such as 250–500 mM)
– Up to 8 M urea
Adjust to pH 8.0.
* For optimal protein purification results, it is crucial that the imidazole
concentrations in lysis, binding, and wash as well as elution buffers be
empirically determined. Optimized conditions should be determined
using a small amount of sample and a 1 ml IMAC column.
Reagents for on-column refolding
• Refolding buffer
–
20 mM imidazole
– 300 mM NaCl
– 1 mM
β-mercaptoethanol
– 20 mM sodium phosphate (NaH
2
PO
4
) (pH 8.0)
• Phosphate elution buffer (with high imidazole)
–
500 mM imidazole
– 300 mM NaCl
– 1 mM
β-mercaptoethanol
– 20 mM sodium phosphate (NaH
2
PO
4
) (pH 8.0)
• Urea binding buffer** (refolding buffer with 8 M urea)
Prepare the urea binding buffer by first weighing out the urea. Add
2 M imidazole, 1 M sodium phosphate (pH 8.0), and water to
approximately 90% of the final volume into a container with a stirbar.
Place the container into a water bath at 45°C as addition of the
denaturant causes the solution to cool. Use caution when warming
urea solutions. Breakdown occurs rapidly above 28°C.
Stir the solution to dissolve the denaturant. Bring the solution to room
temperature before adjusting the pH. Adjust the pH, add the
β-mercaptoethanol, and add water to the final volume. Filter the solution.
Note: The solution must be room temperature before pH adjustment and
heat causes pH to fluctuate. The
β-mercaptoethanol should only be
added to the solution immediately before use.
** The urea binding buffer may be used to wash and elute proteins
prepared with 6 M guanidine HCl.
Equipment
• Chromatography system with dual pump and gradient capability
• IMAC column (as prepared in Section 4)
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