Biology Reference
In-Depth Information
C in 20% FCS, 10% DMSO, 70%
medium. Viral stocks that become contaminated can be sterile-filtered (0.22
important viruses should be frozen at -120
°
µ
M
pore) and retitered.
12.
Characteristic changes in cell morphology are noticeable upon baculovirus infec-
tion. When viewed by phase microscopy, the nucleus tends to be larger and more
prominent in infected cells, and at later stages of infection occupies most of the
intracellular space. It is recommended to compare cell and nuclear morphology
to an uninfected Sf9 control to notice sometimes subtle differences. Cell division
also ceases after infection, and a comparison of cell number with that of
uninfected controls can also be used to as a sign of infection.
13.
Alternatively, recombinant baculovirus can be mixed with cells in suspension
and plated directly.
14.
To insure that your recombinant baculovirus is indeed clonal, repeat dilutional
cloning again (preferably twice) using the single positive well.
15.
As is observed for the growth of primary explants or transformed cell lines in
culture, viruses appear to diverge from the original clone from which they were
derived with increasing passage/expansion in culture. To prevent problems that
may arise upon prolonged usage, we strongly recommend documenting each
passage number. If and when problems begin to become apparent, return to the
earliest possible passage to re-establish baculoviral stocks.
16.
Care should be taken when decanting the supernatant. A low G-force spin pellets
cells without damaging membranes and prevents the release of intracellular protein-
ases. Therefore, the pellet will not be a compact aggregate and be relatively loose.
17.
Equilibration rates are directly proportional to tubing diameter; large-diameter
dialysis tubing requires more time than small-diameter tubing to reach equilib-
rium. In the initial purification steps we use NH
4
HCO
3
so that later fractions can
be rapidly lyophilized and recombinant followed by SDS-PAGE and Western
blotting. Substitution of 50 m
M
Tris pH 8.0, which provides better overall buff-
ering capacity, can be substituted once several large-scale purifications have been
successful.
18.
Be sure to regenerate any column prior to use with buffer containing 1
M
NaCl,
or as recommended by the manufacturer. This removes any residual protein that
may have remained on the column after previous runs and allows optimal bind-
ing capacity for recombinant.
19.
For Tris buffered reagents, pH is
inversely
proportional to temperature. As a general
rule, for each
C the pH changes approx 0.03 pH units. For example, for prepara-
tion of a Tris buffer at room temperature (25
°
°
C) that will ultimately be used at in
the cold (4
0.03 pH units = 0.63 pH
units that the buffer will increase upon decreasing temperature. So, if pH 7.4 is
required at 4
°
C), the difference in temperature is 21
°
C: 21
×
°
C, at room temperature the pH should be adjusted to 6.77.
20.
Buffers used for HPLC should always be filtered and de-gassed to prevent column
clogging and the formation of air bubbles in the column matrix, leading to an
increase in the lifetime of the column.
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