Affinity Chromatography - Department of Molecular and Cellular ...

Purification options
Binding capacity Maximum Comments
operating flow
HiTrap Biotin, > 300 nmol/column 4 ml/min Prepacked 1 ml column.
Streptavidin HP Biotinylated BSA, 6 mg/column
Streptavidin Biotin, > 300 nmol/medium 150 cm/h* Supplied as a suspension
Sepharose Biotinylated BSA, 6 mg/medium ready for column packing.
High Performance
*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.
Purification example
A 280 nm
% elution buffer
1.2
100
Sample:
Column:
Binding buffer:
Elution buffer:
Flow:
System:
9.0 ml of a mixture of BSA and iminobiotinylated
BSA, filtered through a 0.45 µm filter
HiTrap Streptavidin HP, 1 ml
50 mM ammonium carbonate buffer,
0.5 M NaCl, pH 10.0
50 mM ammonium acetate buffer,
0.5 M NaCl, pH 4.0
1 ml/min (0.3 ml/min during
sample application)
ÄKTAexplorer
0.8
0.4
80
60
40
20
0
0
Fig. 39. Purification of iminobiotinylated BSA on HiTrap Streptavidin HP, 1 ml.
0 5 10 15 20 ml
Performing a separation:
Biotinylated substances
Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5
Elution buffer: 8 M guanidine-HCl, pH 1.5
Iminobiotinylated substances
Binding buffer: 50 mM ammonium carbonate, 0.5 M NaCl, pH 10.0
Elution buffer: 50 mM ammonium acetate, 0.5 M NaCl, pH 4.0
1. Equilibrate the column with 10 column volumes of binding buffer.
2. Apply the sample. For the best results use a low flow rate, 0.1–0.5 ml/min, during sample application.
3. Wash with at least 10 column volumes of binding buffer or until no material appears in the eluent (monitored
by UV absorption at A 280 nm ).
4. Elute with 10–20 column volumes of elution buffer.*
*Since elution conditions can be quite harsh, it is recommended to collect fractions into neutralization buffer
(100 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately
neutral or perform a rapid buffer exchange on a desalting column (see page 134).
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