p. 12
Note: If a P1-pump is used a ma x flow rate of 1–3 ml/min can be r un on
a HiTrap 1 ml column packed with S epharose High Per formance
media.
4. OptimizationIf sample compositio n is unknown, a simpl e screening test w ith the aid
of a syringe or pump can be per formed to optim ize starting p H and ionic
strength.
1. Set up a ser ies of buffers wi th different pH ´s, in the range 4–8 (SP ) or 5–9
(Q), with 0.5–1 pH unit int ervals betwee n each buffer. Make one s eries
with 1 M NaCl included in th e buffers (reg eneration buf fer) and the other
without NaCl (start buffer).
2. Equilibrate the column, see Purification.
3. Adjust t he sample to the chos en start buf fer, see Sample prepara tion.
4. Apply a kn own constant amo unt of the sample at 1 or 5 m l/min for the
1 ml and 5 ml columns respec tively. Collec t eluate.
5. Wash wit h at least 5 column vol umes start buf fer or until no mate rial
appears in effluent. Collect eluate.
6. Elute bou nd material wit h elution buffe r. 3–5 column volumes is usu ally
sufficient. Ot her volumes may be re quired, dependi ng on the chosen
operational condit ions. Collec t eluate.
7. Analy ze all eluates for exa mple by activit y assay and SDS-PAGE and
determine the purity a nd the amount bound t o the column.
8. Perfo rm steps 2–7 for the next bu ffer pH.
9. Decide w hich pH should be use d for the selecte d purificatio n strategy.
10. To decide on star ting ionic stre ngth condition s, a similar screeni ng is
done, but the buffer pH i s held constant and t he ionic streng th is varied
in the interval 0–0. 5 M, with interval s of 0.05 to 0.1 M salt betwe en each
buffer.