Biology Reference
In-Depth Information
11.1.5
Purification of TBCE
11.1.5.1
Preparation of insect cell lysates
a.
Dislodge cells that remain attached to dishes containing infected sf9 cells with a
polypropylene scraper. Pool the suspensions into 500 ml conical polypropylene
centrifuge bottles and centrifuge at 2000
g
in a Beckman J6 centrifuge or
equivalent. Remove the supernatant by aspiration with a Pasteur pipet linked to
a vacuum flask, taking care not to disturb the cell pellet.
b.
Resuspend the cell pellet by gently tapping the centrifuge bottle.
c.
Gently add 50 ml of cold T
10
E
1
, swirl to generate a homogeneous suspension,
centrifuge at 2000
g
at 4
C, and remove and discard the supernatant as before.
d.
Resuspend the cell pellet by gently tapping the centrifuge bottle.
e.
Add 5 ml of ice-cold T
10
E
1
containing protease inhibitor cocktail (Roche Inc.).
f.
Transfer to a 15-ml glass Dounce homogenizer with a type A (tight fitting)
pestle. Lyse the cells with 20 full strokes of the homogenizer at 0
C.
g.
Centrifuge the lysate at 500
g
at 4
C for 5 min. Carefully aspirate the
supernatant and transfer to a clean centrifuge tube.
h.
Resuspend the nuclear pellet in half the original volume of ice-cold T
10
E
1
by
persistent tapping, centrifuge at 500
g
as before, and combine the supernatant
with that obtained in step g.
i.
Centrifuge the combined supernatants at 200,000
g
at 4
C for 15 min in an
ultracentrifuge (such as a Beckman Optima).
j.
Collect the supernatants and store flash frozen in liquid nitrogen as described in
Section 1.2.2
c.
11.1.5.2
Anion exchange chromatography
a.
Thaw the flash frozen sf9 cell extracts prepared as described in
Section 1.5.1
in a
37
C water bath, taking care that the melt does not warm above 4
C.
b.
Filter through a 0.4-
Millipore membrane to ensure removal of any aggregated
material that may have formed.
c.
Measure the total protein content of the sample using, for example, the Bio-Rad
Protein Assay Reagent. The total soluble protein recovered from 15 to 20 dishes
(20 cm
m
20 cm) of infected sf9 cells should be in the range of 80-100 mg.
d.
Apply the mixture to an FPLC column of Q High-Performance anion exchange
resin (GE Healthcare Inc.) (1 cm
10 cm) equilibrated in 10 mM Tris-HCl, pH
8.0, 1 mM EDTA, and 1 mM DTT. For volumes in the range of 5-10 ml, it is
most convenient to use the 10 ml superloop for this purpose.
e.
Wash the column after sample loading with 2 column volumes of equilibration
buffer and develop with a 100-ml linear gradient of equilibration buffer
containing 0.25 M NaCl, collecting fractions of 2 ml. The column may be run at
room temperature, but it is advisable to transfer the fractions to an ice bucket as
soon as they emerge from the column.
f.
Analyze a small proportion (e.g., 5
m
l) of the relevant fractions by SDS-PAGE. As a
guide, TBCE emerges from the column in the conductivity range 7.3-10.6 mS/cm.
