Biology Reference
In-Depth Information
8. Clean the column every three runs. CIM
®
Q disks can be
regenerated by submerging the disk in 1 M NaOH for 2 h.
Piston regeneration (the frit regeneration actually) is as fol-
lows: 5 min in 1 M NaOH, 5 min in Milli-Q H
2
O, 5 min in
100 % PrOH, 5 min in Milli-Q H
2
O, and 5 min in a sonicator
(ultrasonic bath).
9. Store the disks in storage buffer at 4 °C and purified samples at
−80 °C (
see
Notes 24
-
26
).
4
Notes
1. The UNICORN system control software associated with the
ÄKTA explorer 100 enables on-line monitoring and control of
the chromatography process.
2. All buffers should be filtered and degassed prior to
chromatography.
3. Fractogel
®
matrices are very spongy. Do not let these matrices
sit in just water as the spacer arms will start clumping together
which would seriously hamper the column's ability to capture
viral particles. Therefore, even the storage buffer should have
at least 0.15 M NaCl in it.
4. Various size CIM
®
DEAE monolithic columns are commer-
cially available for dealing with larger volumes of semi-purified
CAV-2 stocks. This protocol describes the elution strategy that
was originally developed using 0.34 mL CIM
®
DEAE mono-
lithic disks [
16
].
5. While most CAV-2 particles remain located inside the cell after
production, some virus particles may escape to the supernatant
fraction. The amount of CAV-2 particles that can be found in
the supernatant will vary depending on the specific production
procedure employed and should be determined case-by-case.
Using our production protocol, 30 % of the active CAV-2 par-
ticles are located in the extracellular fraction at the time of vec-
tor harvest [
16
]. The latter can easily be recovered by processing
both the extracellular along with the intracellular cell culture
fraction.
6. The media used for CAV-2 production should not contain
phenol red. Viral stocks are produced in the absence of this pH
indicator.
7. Install a pressure gauge inline between the pump outlet and
filter inlet to monitor filter inlet pressure at all times. Use appro-
priate fittings and secure connections. Ensure that the capsule
is installed in the correct direction according to the arrow on
the capsule label. Check all system tubing to ensure it is not
kinked or pinched.
