p. 12
Note: If a P1-pump is used a max flow rate of 1–3 ml/min can be run on
a HiTrap 1 ml column packed with Sepharose High Performance
media.
4. Optimization
If sample composition is unknown, a simple screening test with the aid
of a syringe or pump can be performed to optimize starting pH and ionic
strength.
1. Set up a series of buffers with different pH´s, in the range 4–8 (SP) or 5–9
(Q), with 0.5–1 pH unit intervals between each buffer. Make one series
with 1 M NaCl included in the buffers (regeneration buffer) and the other
without NaCl (start buffer).
2. Equilibrate the column, see Purification.
3. Adjust the sample to the chosen start buffer, see Sample preparation.
4. Apply a known constant amount of the sample at 1 or 5 ml/min for the
1 ml and 5 ml columns respectively. Collect eluate.
5. Wash with at least 5 column volumes start buffer or until no material
appears in effluent. Collect eluate.
6. Elute bound material with elution buffer. 3–5 column volumes is usually
sufficient. Other volumes may be required, depending on the chosen
operational conditions. Collect eluate.
7. Analyze all eluates for example by activity assay and SDS-PAGE and
determine the purity and the amount bound to the column.
8. Perform steps 2–7 for the next buffer pH.
9. Decide which pH should be used for the selected purification strategy.
10. To decide on starting ionic strength conditions, a similar screening is
done, but the buffer pH is held constant and the ionic strength is varied
in the interval 0–0.5 M, with intervals of 0.05 to 0.1 M salt between each
buffer.
