Bio-Rad Profinity IMAC Resins - Manual (Page 9)

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Section 3
General IMAC Procedures
Protein Binding
Protein adsorption to immobilized ions is performed around neutral to slightly
alkaline pH conditions (pH 7.0–8.5). To reduce nonspecific ionic effects,
concentrations of up to 1 M NaCl may be added to the binding solution.
Recombinant 6xHis tags, located at either the amino or carboxyl terminus of the
protein, can bind with high affinity to the matrix even when the 6xHis tag isn’t
completely accessible. In general, the fewer the number of accessible histidine
residues, the weaker the protein binding is to the affinity matrix. Untagged proteins
that have naturally occurring and noncontiguous histidine residues also bind to
IMAC resins, but with much lower affinity.
Batch mode binding is a good alternative if proteins are expressed at low levels
or if the overall concentration of the recombinant 6xHis tag is low. In this case,
proteins are bound to the Profinity
™ IMAC resin in solution prior to packing the
protein-resin complex into a liquid chromatography column for wash and elution
steps. Altering the imidazole concentration of the lysis buffer may also optimize
binding. Low concentrations such as 0–20 mM imidazole are recommended and
will aid in reducing nonspecific binding of weakly interacting proteins.
Many additives can be used without affecting the binding of His-tagged proteins to
IMAC resins, for example, urea, GuHCl, nonionic detergents, and organic solvents
(refer to Section 2, Table 2). Chelating agents, such as EDTA or citrate, should not
be included. Though not recommended, reducing agents such as
β-mercapto-
ethanol and DTT may be used at low concentrations.
Potassium phosphate or sodium phosphate buffers are recommended solutions for
equilibration and binding.
Recommended binding buffer
• 20–50 mM sodium or potassium phosphate, containing up to 1.0 M NaCl,
e.g., 50 mM sodium phosphate, 0.3 M NaCl, pH 8.0
Washes
Stringency of 6xHis tag binding can be effectively increased by 1) including low
concentrations of imidazole in the binding and wash solutions, or 2) reducing the
pH. Generally, highly expressed proteins, such as those expressed using a
bacterial expression system, have fewer contaminant proteins that copurify along
with the protein of interest. Abundance of endogenous protein contaminants is
higher in eukaryotic expression systems and tends to bind to the IMAC adsorbent
more weakly. In these instances, nonspecific binding of proteins containing
neighboring histidine residues becomes a problem. These endogenous species
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