Bio-Rad Profinity IMAC Resins - Manual (Page 10)

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may be washed from the resin by either lowering the pH to 6.3 or adding imidazole
to binding and wash solutions in concentrations of 5–20 mM. The optimal pH
and/or imidazole concentration used in wash buffers is always protein-dependent
and should always be determined experimentally.
Elution
Desorption of the His-tagged protein may be accomplished in one of three ways:
introduction of a competitor ligand, reduction of the pH, or stripping of the
immobilized metal.
In competitive elution, a step or gradient elution with ligands such as imidazole,
histidine, histamine, or glycine may be carried out. When using a gradient elution
with imidazole, it is important to ensure that the column has been preequilibrated
with low concentrations of imidazole (that is, 1 mM) with the same concentration
being included in the sample. This action avoids a drop in pH from occurring
(caused by significant adsorption of imidazole onto the resin), which might
prematurely elute bound His-tagged proteins.
Lowering the pH of the elution buffer (pH 4.5–5.3) also releases bound His-tagged
proteins. In this case, the histidine residues become protonated and are unable to
bind to the immobilized ion. Protein sensitivity to low pH ranges, however, must be
taken into consideration. If lower pH is used to elute bound proteins, tubes filled
with a strong neutral buffer such as 1 M Tris-HCl, pH 8.0 may be used to collect
acidic eluates (that is, 100–200 µl/ml eluate). The recommended range is pH 3–5
with acetate buffers being a preferred choice. Weakly bound contaminants may be
washed off in an intermediate wash at around pH 5.5–6.5.
Strong chelating agents such as EDTA and EGTA strip immobilized ions from the
column and cause the bound His-tagged protein to elute as a protein-metal
complex. This results in metal ions appearing in the protein fractions.
Recommended elution buffer for Profinity IMAC resin
Though a number of conditions can be used to elute the target protein from the
Profinity resin, adjusting the concentration of imidazole is recommended.
• 20–500 mM imidazole, e.g., 50 mM sodium phosphate, 0.3 M NaCl,
0.5 M imidazole, pH 8.0
Purification Under Denaturing Conditions
When overexpressed in E. coli, some proteins may aggregate, forming what are
known as inclusion bodies. These inclusion bodies need to be solubilized in strong
denaturants such as 6 M guanidine HCl or 8 M urea in order to purify the His-
tagged protein. Usually proteins expressed as inclusion bodies are not in their
native conformation, thereby allowing the use of high concentrations of
denaturants during the preparation of lysate and protein purification.
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