Affinity Chromatography - Department of Molecular and Cellular ...

Ligand and column preparation
1. Dissolve the ligand in the coupling buffer to a final concentration of 0.5–10 mg/ml (for protein ligands) or
perform a buffer exchange using a desalting column (see page 134). The optimal concentration depends on
the ligand. Dissolve the ligand in one column volume of buffer.
2. Remove the top cap from the column and apply a drop of ice-cold 1 mM HCl to the top of the column to avoid
air bubbles.
3. Connect the top of the column to the syringe or pump.
4. Remove the twist-off end.
Ligand coupling
1. Wash out the isopropanol with 3 x 2 column volumes of ice-cold 1 mM HCl.
2. Inject one column volume of ligand solution onto the column.
3. Seal the column. Leave for 15–30 minutes at +25 °C (or 4 hours at +4 °C).
Re-circulate the solution if larger volumes of ligand solution are used. For example, when
using a syringe, connect a second syringe to the outlet of the column and gently pump the
solution back and forth for 15–30 minutes or, if using a peristaltic pump, circulate the
ligand solution through the column.
Do not use excessive flow rates (maximum recommended flow rates are 1 ml/min (equivalent
to approximately 30 drops/min when using a syringe) with HiTrap 1 ml and 5 ml/min
(equivalent to approximately 120 drops/min when using a syringe) with HiTrap 5 ml).
The column contents can be irreversibly compressed.
Measure the efficiency of protein ligand by comparing the A 280 values of the ligand solution
before and after coupling. Note that the N-hydroxy-succinimide, released during the
coupling procedure, absorbs strongly at 280 nm and should be removed from the used
coupling solution before measuring the concentration of the remaining ligand. Use a small
desalting column (see page 134) to remove N-hydroxy-succinimide from protein ligands.
Alternative methods for the measurement of coupling efficiency are described on page 103
and in the HiTrap NHS-activated HP instructions.
Washing and deactivation
This procedure deactivates any excess active groups that have not coupled to the ligand and
washes out non-specifically bound ligands.
1. Inject 3 x 2 column volumes of blocking buffer.
2. Inject 3 x 2 column volumes of wash buffer.
3. Inject 3 x 2 column volumes of blocking buffer.
4. Let the column stand for 15–30 min.
5. Inject 3 x 2 column volumes of wash buffer.
6. Inject 3 x 2 column volumes of blocking buffer.
7. Inject 3 x 2 column volumes of wash buffer.
8. Inject 2–5 column volumes of a buffer with neutral pH.
The column is now ready for use.
107