Bio-Rad Profinity IMAC Resins - Manual (Page 31)

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Section 11
Medium-Pressure Column Purification of
His-Tagged Proteins Using Denaturing
Conditions
This guideline uses denaturants such as urea to solubilize inclusion bodies, which
are not generally in their native conformation. Elution is achieved by increasing the
imidazole concentration. The protocol also recommends how to restore the protein
to its native form. In this case, the denaturant used to lyse and purify the sample
must be removed using dilution, dialysis, or size exclusion chromatography.
The guideline does not optimize the imidazole concentration, but instead allows
fast capture of the target protein and may be used as a quick check for protein
expression levels.
Renaturation of the protein while it is still bound to the IMAC column is a good
alternative and offers several advantages. Aggregation may be kept to a minimum
if the protein refolds on the column when the denaturant is removed. Higher
concentrations of the refolded protein may therefore be collected. Finally, with the
use of a liquid chromatography system, denaturing agents, detergents, salts, and
pH can be adjusted and effectively controlled.
Note: If using guanidine HCl (GuHCl), it must be removed from purified samples
prior to loading onto SDS-PAGE gels due to precipitation that occurs. Proteins that
have been lysed and adsorbed onto the column with guanidine HCl may be
washed and eluted with a urea-based buffer.
A gradient elution protocol (see Section 10) may be used with small sample
volumes to optimize the imidazole concentration needed in the stepwise elution
protocol.
Materials
Reagents for Protein Binding and Elution
• Binding buffer (urea-based)
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– Low concentration of imidazole* (such as 0–10 mM)
– Up to 8 M urea
Adjust to pH 8.0.
• Wash buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– Low concentration of imidazole* (such as 0–20 mM)
– Up to 8 M urea
Adjust to pH 8.0.
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