View - ResearchGate

130 Forde
4. Wash the column with 5 column volumes of PBS buffer at a flow rate of 60 cm/h
or until the optical density at 280 nm (OD 280 nm ) of the column outlet stream
returns to base-line levels.
5. Elute the GST–ZnF protein using a solution of 20 mM reduced glutathione (see
Note 15), pH 9 (see Note 16), and 100 mM Tris-HCl for buffering at a flow rate
of 60 cm/h.
6. Remove excess glutathione present in the elution fraction by dialysis. Dialysis
tubing with a molecular weight cut-off of 12,000 Daltons is suitable. Autoclave
the dialysis tubing. Secure one end of the dialysis tubing so that it is water tight
(i.e., do not allow the passage of liquids out of one end), load the elution solution
into the open end of the dialysis tubing, then secure the second end.
7. Place the loaded tubing into 4°C PBS buffer and stir using a magnetic bar stirrer
at 100 rpm. Ensure that the temperature is maintained at 4°C. Approximately 500
ml of PBS buffer should be used per 1 ml of elution fraction. It is recommended
that dialysis be performed for a period of at least 24 h. Exchanging the dialysis
buffer with fresh PBS buffer enables faster removal of glutathione from the elution
solution.
3.7. Expanded Bed Adsorption
1. Load the chromatography column via gravity settling with the adsorbent prepared
as given in Subheading 3.1.
2. Equilibrate the column with at least 10 settled bed column volumes of PBS buffer
using upward flow to expand the column. Expand the bed to twice its settled bed
height. In a 1-cm diameter column, use a flow rate of approximately 150 cm/h
(see Note 17).
3. Using upward flow, pump GST–ZnF-containing lysate into the column at
150 cm/h.
4. Some column expansion is to be expected due to the higher density and viscosity
of the feed. To prevent loss of adsorbent through the top of the column, the flow
may need to be reduced or the position of the top column frit adjusted.
5. Wash the column with 5 settled column volumes of PBS buffer at 150 cm/h or
until the optical density at 280 nm (OD 280 nm ) of the column outlet stream returns
to base-line levels.
6. Reverse the flow of PBS buffer to downward flow and lower the top adaptor
in order to operate the column in packed bed mode (see Note 18). Continue
downward flow at 150 cm/h with PBS buffer until the optical density at 280 nm
(OD 280 nm ) of the column outlet stream is at base-line levels.
7. Elute the GST–ZnF in packed bed mode at 150 cm/h with 20 mM reduced
glutathione, pH 9, 100 mM Tris–HCl with approximately 3 settled column
volumes. Refer to the optical density at 280 nm (OD 280 nm ) to monitor the
elution peak.