Bio-Rad Macro-Prep 25 S Media - Manual (Page 9)

7
Sample Preparation
Adjust salt concentration and pH as necessary to achieve desired selectivity for
binding of target or contaminants. If sample conductivity exceeds conductivity of
the column equilibration buffer, sample capacity may be reduced or the target
molecule may not bind. Adjustment of pH and conductivity of sample can be done
by dilution or buffer exchange.
Sample Load and Adsorption
The sample load is determined empirically by loading and evaluating breakthrough
of the molecule of interest. Sample volume is not a critical factor. Large volumes of
dilute feed such as cell culture supernatant and clarified lysates may be loaded
onto the supports without prior concentration.
Wash-Through
When operating in a bind-elute mode, after loading of the sample onto the
column, follow with 2–5 CV of the equilibration buffer. This will wash out unbound
contaminants. In flow-through mode, after sample is completely through the
column, regenerate the resin as described under “Regeneration” below.
Elution
Elute target molecules with a step or a linear gradient. The salt concentration at
which the desired product elutes is predetermined at small scale. With this
knowledge, the pH and salt concentration used in wash-through are adjusted to
eliminate the maximum amount of contamination before starting elution of the
target.
Regeneration
After each run, the packed bed should be washed with 2–4 CV of a high salt
buffer (0.5–2.0 M) to remove remaining bound material. Samples may then be
loaded onto the column after reequilibration in starting buffer. If column no longer
yields reproducible results, the supports may require more thorough cleaning and
sanitization.
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