Biology Reference
In-Depth Information
3.4 Chromatography
Purification
1. Turn on the ÄKTA explorer and wash pump A & B with
Milli-Q H
2
O
2. Install the 10-mL Fractogel
®
propyl column and remove the
storage buffer with 10 column volumes (CV) of Milli-Q H
2
O
at a linear flow rate of 92 cm/h (3 mL/min).
3. Wash pump A and B with the corresponding buffers. Monitor
UV absorbance at 280 and 260 nm (
see
Note 16
). Equilibrate the
column with binding buffer (0.85 M NH
4
(SO
4
)
2
in buffer A).
4. Thaw the tenfold concentrated CAV-2 stock. Dilute sample in
buffer A containing 1.7 M NH
4
(SO
4
)
2
to match the conduc-
tivity of the binding buffer (120 mS/cm) (typically 1:2). Filter
the conditioned chromatography feed using a 0.45-μm pore
size syringe-mounted filters. Aliquot starting material for anal-
yses (
see
Note 17
).
5. When stable baseline is achieved, load the conditioned CAV-2
feed (
see
Note 18
) and apply a step-wise gradient elution strat-
egy that includes a wash step in binding buffer (5 CV) to
remove the bulk of contaminating proteins, followed by CAV-2
elution with buffer A using a single-step gradient (16 CV) (
see
Note 19
). The process is carried out at room temperature at
153 cm/h (5 mL/min).
6. The virus particles elute in a defined peak (
see
Fig.
3a
). Pool
virus-containing fractions and aliquot for analyses (
see
Note 20
).
7. After each run, wash the column with buffer A and re-
equilibrate the column with binding buffer containing 0.85 M
NH
4
(SO
4
).
8. Clean the column with 0.5 M NaOH every three runs. Load
on at least 3 CV of the cleaning solution and let sit for at least
20 min. NaOH can be removed by rinsing the column with 10
CV of 0.15 M NaCl in Milli-Q H
2
O. Re-equilibrate with at
least 10 CV of the equilibration buffer. Check pH at column
outlet prior to the next run.
9. Store the column in storage buffer with 10 CV at 92 cm/h.
10. Keep semi-purified samples at 4 °C until further use.
3.4.1 CAV-2 Capture by
Hydrophobic Interaction
Chromatography
1. Turn on the ÄKTA explorer and wash pump A & B with
Milli-Q H
2
O
2. Install the CIM
®
DEAE monolithic disk and remove the stor-
age buffer with 10 CV of Milli-Q H
2
O at a flow rate of 2 mL/
min.
3. Wash pump A and B with the corresponding buffers. Monitor
UV absorbance at 280 and 260 nm. Equilibrate the column
with binding buffer to attain a stable baseline. Binding buffer
consists of 160 mM NaCl in buffer A.
3.4.2 Polishing of CAV-2
Using Anion Exchange
Chromatography Monoliths
