p. 11
Purifi cation 1. Fill th e syringe or pump tub ing with start b uffer (low ion ic strength) .
Remove the stopper and c onnect the colum n to the syringe (wi th the
provided adaptor), “dr op to drop” to avoid intro ducting air into t he
column.
2. Remove t he snap-off en d at the column outle t.
3. Wash out t he preservati ves with 5 column volu mes of start buf fer, at
1 ml/min for the 1 ml column an d 5 ml/min for the 5 ml col umn.
4. Wash wit h 5 column volumes of el ution buffer ( start buff er with
1 M NaCl).
5. Finally equilibrate with 5–10 column volumes of start buffer.
6. Apply t he sample at 1 or 5 ml/m in for the 1 ml and 5 ml colu mns
respectively, usi ng a syringe fit ted to the luer adapto r or by pumping it
onto the column.
7. Wash wit h at least 5 column vo lumes of start bu ffer or until no ma terial
appears in the eff luent.
8. Elute wi th 5–10 column volumes of elu tion buffer (s ee section “Cho ice of
gradient type”).
9. The pur ified fracti ons can be desalte d using a HiTrap Desalt ing,
HiPrep 26/10 Desalting or a PD-10 column s if necessary.
10. After the co mpleted elution , regenerate the co lumn by washing wit h
5 column volumes of regene ration buffer ( start buf fer with 1 M NaCl)
followed by 5–10 column volumes o f start buffer. Th e column is now
ready for a new sample.
For a first experim ent the following co nditions are reco mmended:
Flow rate: 1 ml/min using HiTrap 1 ml column
5 ml/min using HiTrap 5 ml column
Start buffer: See Table s 2 and 3
Elution buffer: Start buffer + 1 M NaCl
Gradient volume: 20 ml