Bio-Rad Macro-Prep 25 S Media - Manual (Page 7)

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7. Switch off the pump and close the column outlet. Disconnect the flow
adapter tubing from the pump and position the flow adapter in contact with
the support. During this step, the packing solution will backflow through the
adapter.
8. Reconnect the flow adapter tubing to the pump, open the column outlet, and
run 4–5 more CV of 0.5–1.0 M NaCl at the highest flow rate your pump/
column system will permit, then repeat steps 7 and 8 for a final adjustment of
the flow adaptor.
9. After equilibration in the start buffer, the column is ready for sample
application.
Section 6
Operation and Maintenance
Macro-Prep 25 ion exchange columns may be operated in two different modes.
In bind-elute mode, the protein of interest (target molecule) binds to the support
during loading. It is subsequently eluted by a change of conductivity and/or pH.
This allows removal of contaminants that do not bind to the support and
contaminants that may bind more weakly or more strongly than the protein of
interest. In flow-through mode, the protein of interest does not bind during loading,
while contaminants that may bind to the support are removed. This approach can
simplify chromatography conditions, conserve buffer, and increase recovery of the
target molecule.
For flow-through mode, a linear flow rate of 300 cm/hr in a 20 cm bed is practical.
If operating in a bind-elute mode, flow rates of 100–200 cm/hr are recommended.
Do not exceed 70–75% of the maximum pressure attained during the initial
column packing. If you do not have a pressure indicator, do not exceed flow rates
in excess of 70–75% of the maximum flow rate used during packing.
All buffers used for anion or cation exchange chromatography can be used with
the appropriate ion exchange supports (see Table 2). It is best to use buffering
ions that have the same charge as the functional group on the ion exchanger, for
example, phosphate (–) with the Macro-Prep 25 S cation exchanger and Tris (+)
with the Macro-Prep 25 Q anion exchanger. Buffers should be chosen so
operating pH is within 0.5 pH unit from pK
a
of buffer substance.
The purification may be optimized by changing the pH, ionic strength of elution
buffer, gradient profile, or buffer. Typical chromatographic conditions are use of
buffer concentrations in the range of 20–50 mM, at least one pH unit below the pI
of the protein for cation exchange, and one pH unit above the pI of the protein for
anion exchange. Gradients are then run from 0 to 1 M NaCl over 10–20 CV.
Proper adjustment of the sample pH and ionic strength is critical for consistent
and reproducible chromatography. For best results, the sample should be
exchanged into the start buffer or diluted to the concentration of the start buffer.
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