Affinity Chromatography - Department of Molecular and Cellular ...

Performing a separation
Binding buffer: 140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.3
Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0
A B C
Fig. 23. Using GSTrap FF with a syringe. A: Prepare buffers and sample. Remove the column’s top cap and twist off the
end. B: Equilibrate column, load the sample and begin collecting fractions. C: Wash and elute, continuing to collect
fractions.
1. Equilibrate the column with 5 column volumes of binding buffer.
2. Apply the sample.
3. Wash with 5–10 column volumes of binding buffer.
4. Elute with 5–10 column volumes of elution buffer.
5. Wash with 5–10 column volumes of binding buffer.
To improve yield try decreasing the flow rate or passing the sample through the column
several times.
For a single purification of a small quantity of product or for high throughput screening,
GST MicroSpin columns are convenient and simple to use with either centrifugation or
MicroPlex 24 Vacuum.
To increase capacity, connect several GSTrap FF columns (1 ml or 5 ml) in series or, for
even larger capacity, pack Glutathione Sepharose 4 Fast Flow into a suitable column (see
Appendix 3). GSTrap FF columns can be used with a syringe, a peristaltic pump or a
chromatography system.
Enzyme-specific recognition sites are often included to allow the removal of the GST tag by
enzymatic cleavage when required. Thrombin is commonly used for enzymatic cleavage,
and must, subsequently, be removed from the recombinant product. HiTrap Benzamidine FF
(high sub) 1 ml or 5 ml columns provide simple, ready-made solutions for this process
(see page 53).
Reuse of GSTrap FF depends on the nature of the sample. To prevent cross-contamination,
columns should only be reused with identical samples.
44