Biology Reference
In-Depth Information
5. Cleaning solutions: 0.5 M NaOH, 20 % EtOH, Milli-Q H
2
O.
6. Storage solution: 20 % EtOH.
1. Benzonase grade I 25,000 U (Merck Millipore, Darmstadt,
Germany).
2. Digestion buffer: 100 mM Tris-HCl, 100 mM MgCl
2
, pH 8
filter-sterilized buffer.
2.3 Benzonase
®
Treatment
2.4 Chromatography
Purification
1. Low-pressure liquid chromatography system (AKTA explorer
100; GE Healthcare) equipped with UV, conductivity and pH
meters (
see
Note 1
).
2. Fractogel
®
EMD propyl gel (S) (Merck Millipore) packed into
a XK 16/20 column (GE Healthcare) to a final volume of
10-mL.
3. 0.45 μm pore size Millex-HV PVDF syringe-mounted filters
(Merck Millipore).
4. Buffer A: 20 mM Tris-HCl, 2 mM MgCl
2
, 10 mM NaCl,
2.5 % glycerol, pH 8 (
see
Note 2
).
5. Buffer B: 2 M NH
4
(SO
4
)
2
in 20 mM Tris-HCl, 2 mM MgCl
2
,
10 mM NaCl, 2.5 % glycerol, pH 8.
6. Storage solution: 20 % EtOH + 150 mM NaCl in Milli-Q H
2
O
(
see
Note 3
).
7. Cleaning solution: 0.5 M NaOH.
2.4.1 CAV-2 Capture by
Hydrophobic Interaction
Chromatography
1. Low-pressure liquid chromatography system (AKTA explorer
100; GE Healthcare) equipped with UV, conductivity and pH
meters.
2. Convective Interaction Media (CIM
®
) DEAE monolithic disks
(0.34 mL) (Bia Separations, Ljubljana, Slovenia) (
see
Note 4
).
3. Buffer A: 20 mM Tris-HCl, 2 mM MgCl
2
, 10 mM NaCl,
2.5 % glycerol, pH 7.
4. Buffer B: 2 M NaCl in 20 mM Tris-HCl, 2 mM MgCl
2
,
10 mM NaCl, 2.5 % glycerol, pH 7.
5. Storage solution: 20 % EtOH.
6. Cleaning solution: 1 M NaOH.
2.4.2 Polishing of CAV-2
Using Anion Exchange
Chromatography Monoliths
3
Methods
3.1 Clarification
by Dead-End
Microfiltration
1. Thaw CAV-2 virus stocks using water bath at 37 °C (
see
Notes
5
and
6
).
2. Aliquot starting material samples for analyses and measure the
starting volume. Keep the viral stock at 4 °C until further
processing.
