p. 15
– A peristaltic pump and a gradien t mixer e.g. pump P-1, gradient m ixer
GM-1.
– A one pump system, e.g. ÄKTAprime™ plus.
– A two pump system, e.g FPLC or ÄKTA.
1. Choose starting conditions as outlined under Optimizing starting
conditions.
2. Equilibrate the column, see Purification.
3. Adjust t he sample to the chos en starting pH a nd ionic strengt h, see
Sample preparation.
4. Apply t he sample at 1 or 5 ml/mi n for the HiTrap 1 or 5 ml colum n
respectively. Collect eluate.
5. Wash wit h 5–10 column volumes of star t buffer or until n o material
appears in effluent.
6. Start t he gradient eluti on. A gradient volum e of 10–20 column volumes
and an increase in ionic s trength to 0.5 M NaCl i s usually suffi cient.
7. Regene rate the column by was hing with 5 column vo lumes of start
buffer with 1 M NaCl foll owed by 5–10 column volumes of s tart buffer.
The column is now ready for a n ew sample.
6. Determination of binding capacity
The amount of sample whic h can be applied to a colu mn depends on the
capacity of the column and t he degree of resolu tion required. T he capacity
is dependent on the sample composition, choosen starting conditions of pH
and ionic strength an d the flow rate at whi ch the separatio n is done. The
influence of flow rate a nd pH on the capacity fo r some model protei ns are
shown in Figure 4.
Samples were applied u ntil 5% of the start m aterial appear ed in the eluent.
The column was then wash ed with 10 ml start bu ffer (20 mM Tris-HCl, pH 8 .2
or 9.0) before elution wi th elution buf fer (20 mM Tris-HCl, 1.0 M NaCl , pH 8.2
or 9.0).
1. Equilibrate the column, see Purification.
2. Adjust t he sample to the chos en starting pH a nd ionic strengt h, see
Sample preparation.
3. Determi ne the concentrat ion of the specif ic proteins by UV, SDS- PAGE,
ELISA or other appropria te techniques.