Biology Reference
In-Depth Information
Fig. 1
Baculovirus
facilities at UVHCI (Unit of Virus Host Cell Interactions in Grenoble). Insect cell is cultured
under agitation in a 27 °C thermostated room
grow until a concentration of about 10
6
cells per mL is reached.
200 mL of culture can be considered a “standard
production.”
3. Infect cells at a multiplicity of infection “m.o.i” = 4, by adding
the
Baculovirus
aliquot under the sterile hood. After 30 min
incubation, put the spinner back under agitation for 60 h.
4. 60 h later, under the hood, dispatch the infected cells into
50 mL sterile plastic tubes. Equilibrate and centrifuge the
tubes at 600 ×
g
for 5 min at room temperature. Discard the
supernatant and store the pellet immediately at −20 °C (
see
Note 4
).
5. For purifi cation, extemporaneously thaw the pellet by adding
500
L of hypotonic buffer supplemented with protease cock-
tail inhibitor and pool all the pellets together. Perform three
freezing-thaw cycles (−20 °C, +37 °C) and vortex at each
thawing step. Transfer to a 1.5 mL Eppendorf tube, equili-
brate, and centrifuge at maximum speed (13,000 ×
g
) for 5 min
at 4 °C. The cell lysate is then set aside for the next step,
whereas pellets of broken cells and debris are discarded in the
biological trash.
μ
3.2 Purifi cation
and Dialysis
1. Prepare 500 mL of HBS-G and 200 mL of a 50 % Sucrose in
HBS-G. A range of 50 mL solutions containing 15-40 % sucrose
with an interval step of 5 % is then prepared (
see
Note 1
).
2. In order to prepare the sucrose gradient, 1.8 mL of the 40 %
sucrose solution is added to the bottom of two ultraclear
centrifuge tubes. Cushions are then created by gentle addition
of 1.8 mL of the following solutions, starting by the 35 % and
