Bio-Rad Profinity IMAC Resins - Manual (Page 50)

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His-tagged protein is
not pure
Contaminants elute with
target protein
Strongly bound
contaminants elute with
protein
Association of
contaminating proteins with
target protein via disulfide
bonds
Association between the
His-tagged protein and
protein contaminant
Potential degradation of
fusion protein by proteases
Contaminants exhibit
similar affinity to target
protein
Make binding and wash
steps more stringent.
Include 10–20 mM
imidazole in binding and
wash buffers
Prolong the wash step
containing imidazole
Column is too large;
reduce amount of
Profinity
™ IMAC resin used
Very high concentrations
of imidazole will cause
strongly bound
contaminants to elute as
well. Reduce the imidazole
concentration during the
elution
Include ≤30 mM
β-mercaptoethanol. Exert
caution if using DTT
Add nonionic detergent or
alcohol (that is,
≤2% Triton
X-100, 2% Tween 20, or
≤50% glycerol) to reduce
hydrophobic interactions.
Concentration of NaCl
may be increased to
minimize electrostatic
interactions
Include 1 mM PMSF or
other protease inhibitor to
lysis buffer to reduce
partial degradation
Add an additional
chromatography step, that
is, ion exchange,
hydrophobic interaction, or
size exclusion
Problem Possible Cause Solution
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