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p. 11
Purifi cation
1. Fill the syringe or pump tubing with start buffer (low ionic strength).
Remove the stopper and connect the column to the syringe (with the
provided adaptor), “drop to drop” to avoid introducting air into the
column.
2. Remove the snap-off end at the column outlet.
3. Wash out the preservatives with 5 column volumes of start buffer, at
1 ml/min for the 1 ml column and 5 ml/min for the 5 ml column.
4. Wash with 5 column volumes of elution buffer (start buffer with
1 M NaCl).
5. Finally equilibrate with 5–10 column volumes of start buffer.
6. Apply the sample at 1 or 5 ml/min for the 1 ml and 5 ml columns
respectively, using a syringe fitted to the luer adaptor or by pumping it
onto the column.
7. Wash with at least 5 column volumes of start buffer or until no material
appears in the effluent.
8. Elute with 5–10 column volumes of elution buffer (see section “Choice of
gradient type”).
9. The purified fractions can be desalted using a HiTrap Desalting,
HiPrep 26/10 Desalting or a PD-10 columns if necessary.
10. After the completed elution, regenerate the column by washing with
5 column volumes of regeneration buffer (start buffer with 1 M NaCl)
followed by 5–10 column volumes of start buffer. The column is now
ready for a new sample.
For a first experiment the following conditions are recommended:
Flow rate: 1 ml/min using HiTrap 1 ml column
5 ml/min using HiTrap 5 ml column
Start buffer: See Tables 2 and 3
Elution buffer: Start buffer + 1 M NaCl
Gradient volume: 20 ml