Bio-Rad Affi-Gel 15 Gel - Manual (Page 17)

Another method is to titrate the gel with sample,
checking the supernatant for unbound sample after each
addition. This can be done either in a column or in a batch
mode. Continue until the gel is saturated. This method can
be used with a small amount of gel and sample to deter-
mine the capacity and the amount of gel required for the
purification.
9.2 Removal of Unbound Solutes
Proteins or other solutes which are not bound, or are
weakly bound by non-specific interactions, must be
washed off prior to elution. This can be done by washing
with mild chaotropic solutions (1 M NaSCN, 1 M guani-
dine hydrochloride, 1 M urea), 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
which may complicate final elution and helps yield a more
highly purified product.
29
Fig. 5. Use only the required amount of affinity support.
A)
An excess of affinity support is used. During elution, sample
is exposed to excess capacity resulting in great dilution and a
broad peak.
B) The sample is added to the top of the column,
then eluted using reverse flow. Only the required capacity is
used, resulting in minimal dilution and a sharper peak.
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flow
adaptor
elution
saturated gel
excess
capacity
A B
binding
binding
elution
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