Bio-Rad Profinity Epoxide Resin - Manual (Page 9)

7
Section 4
Protein Binding and Elution
General strategies are listed below. Refer to textbooks (Gagnon 1996, Hermanson
et al. 1992, Matejtschuk 1997) and literature articles for additional information and
guidance.
Binding
Use only the required amount of Profinity support. If excess support is used,
sample elution becomes more difficult because the sample continues to bind and
elute as it passes down the column. Stronger elution conditions become
necessary, residence time is longer, the eluted peak is broader, and there is a
greater risk of denaturation and poor recovery. One method to ensure that only the
required amount of Profinity resin is used is to apply the sample to the top of the
column and elute using reverse flow. Another method is to titrate the resin with
sample, checking the supernatant for unbound sample after each addition.
Continue until the resin is saturated. This method can be used with a small
amount of resin and sample to determine the resin capacity and the amount of
resin required for the purification.
Removal of unbound solutes
Proteins or other solutes that are not bound, or are weakly bound by nonspecific
interactions, must be washed off prior to elution. This can be done by washing
with binding or equilibration buffers, with salts (1 M NaCl), or with detergents
(0.5% Triton X-100). In many cases, the elution buffer can be used, but at a lower
concentration. This frequently neglected wash step eliminates proteins that may
complicate final elution and helps yield a more highly purified product.
Elution
Elution is usually the most demanding step in affinity chromatography. Often the
objective is to obtain high purity and high recovery of a stable and active product.
In attempting to maximize yields, elution conditions that denature the proteins are
often chosen.
Antigens and antibodies are bound to each other by a combination of ionic
bonding, hydrogen bonding, and hydrophobic interactions (Frost et al. 1981). The
strength of different antigen-antibody complexes varies widely. Other parameters
such as ligand density, steric orientation, and nonspecific interactions can be
important.
Acid elution is the most commonly employed desorption method and is
frequently very effective. Eluants such as glycine-HCl (pH 2.5), 20 mM HCl, and
sodium citrate (pH 2.5) can be used to disrupt antigen-antibody interactions. Acid
elution can give low recoveries due to hydrophobic interactions between the
antigen and the antibody. An eluant such as 1 M propionic acid, or the addition of
10% ethylene glycol to an acidic eluant, is more effective in dissociating such
complexes.
10003245A.qxd 3/21/2005 10:51 AM Page 7