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62 Gallant et al.
Binding and elution conditions on dye ligand chromatography are a function
of several variables (see Table 1). In some cases, for some proteins and dye
ligands, binding through charge–charge interactions may dominate. In those
cases, binding and elution will most effectively be controlled by varying the
conductivity of the mobile phase (i.e., salt concentration) and by varying the
pH (4). In other cases, the hydrophobic component of the interaction may be
quite strong. In those cases, addition of a solvent or a detergent to the elution
buffer may be required in order to elute the product.
Table 1
Control of Protein Binding and Elution in Dye Ligand Chromatography
Physical variable
Effect
pH
pH exerts a strong affect on binding and elution.
pH for binding may range from 4 to 8 and may
be controlled using sodium acetate (pKa 4.8), MES
(pKa 6.3), phosphate (pKa 7.2), HEPES (pKa 7.6) or
other appropriate buffers. Choosing an alternate buffer
system can sometimes resolve solubility problems and
is worth considering during batch screening (5).
Salts Low ionic strength (typically below 100 mM)
enhances binding to the charged dye ligands.
Extremely low ionic strengths (below 20 mM) can
enhance protein solubility problems. One convenient
means of elution is to increase ionic strength
(100–1000 mM). If 1 M salt is insufficient to elute the
protein, then either the pH must be modified during
elution or solvent or detergent must be added during
elution (see Note 6).
Solvents
Hydrophobic interactions enhance the affinity of dye
ligands for proteins. To increase protein yield, the
elution buffer strength may be enhanced by addition
of non-denaturing solvents such as ethylene glycol or
glycerol. Up to 50% maybe used.
Chaotropic agents
and detergents
Chaotropic agents, such as urea and guanidine, may
be employed to enhance either the elution effect or the
washing affect of a buffer. Non-ionic detergents, such
as Tween 80 and Triton X100, may also be employed
(6). These components modulate the hydrophobic
interactions of proteins with the dye ligand. Care
should be taken to insure that the selected concentration
is compatible with protein activity.