Biology Reference
In-Depth Information
Fig. 3
HIC and AEX chromatography profiles. (
a
) CAV-2 elution profile on Fractogel
®
EMD propyl (S) packed
columns. The concentrated CAV-2 feed (70 mL after conditioning to reach appropriate conductivity) was
loaded onto a 10 mL HIC column equilibrated in 0.85 M NH
4
(SO
4
)
2
buffer. The virus was eluted by step gradi-
ent at 0 M NH
4
(SO
4
)
2
. (
b
) CAV-2 elution profile on CIM
®
DEAE monolithic disks. The semi-purified CAV-2 feed
(3 mL after conditioning to reach appropriate conductivity) was loaded onto a 0.34 mL AEX monolithic disk
equilibrated in 0.16 M NaCl buffer. The virus was eluted by step gradient at 0.34 M NaCl. The
arrow
indicates
CAV-2 elution peak
4. Dilute partially purified CAV-2 samples in buffer A to attain
binding conditions (just under 20 mS/cm) (typically 1:4 or
1:4.5). Aliquot starting material for analyses (
see
Note 21
).
5. When stable baseline is achieved, load the semi-purified virus
sample (
see
Note 22
) and apply a step-wise gradient elution
strategy that includes a wash step in binding buffer (22 CV),
followed by a virus elution step at 340 mM (44 CV) and a final
stringent wash at 1,000 mM NaCl (18 CV). The process is car-
ried out at room temperature at 2 mL/min.
6. Virus particles can be recovered in a peak eluting at 340 mM
(~27 mS/cm) (
see
Fig.
3b
). Pool virus-containing fractions
and aliquot for analyses (
see
Note 23
).
7. After each run, re-equilibrate the column with binding buffer.
