Biology Reference
In-Depth Information
benzonase
®
stock is diluted 1/25 in digestion buffer pH 8 just
prior to use.
15. Optimal digestion conditions have been previously determined
showing over 80 % nucleic acid removal [
16
].
16. Simultaneous monitoring at 260 and 280 nm will help identify
purified virus peaks as purified adenovirus chromatography
peaks typically display a 260/280 absorbance ratio between
1.29 and 1.35 [
20
].
17. It is recommended that samples are not stored at 4 °C for long
periods of time between ultrafiltration and chromatography
steps, as this practice may result in loss of virus particles upon
filtration, presumably due to CAV-2 aggregation.
18. The dynamic capacity of the Fractogel
®
propyl (S) matrix for
CAV-2 particles was determined to be 0.45 × 10
12
vg/mL of gel,
which typically corresponded to 7 mL of conditioned CAV-2
feed. This capacity is comparable to others described in the lit-
erature for adenovirus particles (~0.5 to 5 × 10
12
vp/mL) [
17
].
19. Note that CAV-2 elution from HIC columns typically start as
soon as the conductivity drops below 110 mS/cm.
20. Recovery of CAV-2 in the eluted peak is excellent as deter-
mined by real-time qPCR (88 %,
n
= 9). In addition, a 3.5-fold
concentration of CAV-2 samples in the 10-mL column can be
expected.
21. It is recommended that semi-purified samples are subjected
AEC directly after HIC, without being stored and with no
need for sample microfiltration prior to chromatography. The
latter has been associated with loss of virus particles.
22. The dynamic capacity of CIM
®
DEAE disks for CAV-2 parti-
cles was determined to be 0.70×10
12
vg/mL of monolith,
which typically corresponded to 3 mL of semi-purified condi-
tioned CAV-2 feed per disk [
16
].
23. The majority of virus particles are eluted by step-gradient at
0.34 M NaCl (58 %,
n
= 3) in the first peak eluting at 27 mS/
cm (Fig.
3b
). A second small peak that could not be separated
from the virus elutes during this step at 30 mS/cm. The latter
contains only a small fraction of virus particles and can be col-
lected together with the virus increasing the final yield to
~70 % or separately by appropriate sample fractionation. The
remaining particles are eluted at 42 mS/cm in the high-
stringency wash step at 1 M NaCl (30 %).
24. Due to the weak binding affinity of CAV-2 particles to AEC
matrices under the conditions optimized for chromatography,
purified samples possess neutral pH and low conductivity.
Thus, a subsequent ultrafiltration step to remove salt may not
be necessary unless the virus needs to be further concentrated
