Affinity Chromatography - Department of Molecular and Cellular ...

Purification options
Binding capacity Maximum Comments
operating flow
HiTrap Benzamidine FF Trypsin, > 35 mg/column 4 ml/min (1 ml column) Prepacked columns**.
(high sub) Trypsin, > 175 mg/column 15 ml/min (5 ml column)
Benzamidine Sepharose 4 Trypsin, > 35 mg/ml medium 300 cm/h* Supplied as a
Fast Flow (high sub)
suspension ready for
column packing**.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from
measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
**Supplied in 0.05 M acetate, pH 4 containing 20% ethanol.
Purification examples
Figure 30 shows an example of the removal of trypsin-like proteases from human plasma to
prevent proteolysis of the plasma components, using a low pH elution. The activity test
demonstrated that almost all trypsin-like protease activity is removed from the sample and
bound to the column.
Sample: 1 ml human plasma filtered
through a 0.45 µm filter
Column: HiTrap Benzamidine FF
(high sub), 1 ml
Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl,
pH 7.4
Elution buffer: 50 mM glycine, pH 3.0
0–100% elution buffer in
one step
Flow:
1.0 ml/min
System: ÄKTAexplorer
Protease activity: S-2288 from Chromogenix,
Heamochrom Diagnostica AB
A 405 measurement. The activity
is presented as the proteolytic
activity/mg protein
A 280
IU/litre
A 405
3.0
1.00
A 280
2.5
0.80
2.0
0.60
1.5
0.40
1.0
0.20
0.5
0
0.0 5.0 10.0 15.0 ml
Fig. 30. Removal of trypin-like serine proteases from human plasma using HiTrap Benzamidine FF (high sub), 1 ml.
Figure 31 shows the effectiveness of using a GSTrap FF column with a HiTrap Benzamidine FF
(high sub) for purification of a GST fusion protein, followed by cleavage of the GST tag via
the thrombin cleavage site and subsequent removal of the thrombin enzyme. The GST fusion
protein binds to the GSTrap FF column as other proteins wash through the column. Thrombin
is applied to the column and incubated for 2 hours.
A HiTrap Benzamidine FF (high sub) column, pre-equilibrated in binding buffer, is attached
after the GSTrap FF column and both columns are washed in binding buffer followed by a
high salt buffer. The cleaved protein and thrombin wash through from the GSTrap FF column,
thrombin binds to the HiTrap Benzamidine FF (high sub) column, and the eluted fractions
contain pure cleaved protein.
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