Biomedical Engineering Reference
In-Depth Information
1. Procyclic
T. brucei
cells (
see
Note 3
) are cultivated in
Cunningham's medium supplemented with 15 % heat-
inactivated fetal calf serum at 28 °C, to near stationary phase
(~1.0 × 10
7
/ml). A 300 ml cell culture (~3 × 10
9
cells) is har-
vested by centrifugation at 2,000 ×
g
for 10 min, in a tabletop
centrifuge (Sorvall Legend RT+, Thermo Scientifi c with
swinging buckets).
2. Wash the cells twice with 10 ml PEM buffer.
3. Resuspend the cells in 10 ml PEM buffer containing 1 % NP40
and protease inhibitors. Place on a rotation wheel to facilitate
cell extraction for 15 min.
4. Centrifuge at 3,000 ×
g
for 10 min at 4 °C.
5. Repeat the extraction steps once with 10 ml PEM containing
1 % NP40 and twice with 10 ml PEM containing 1 % NP40
and 1 M KCl. Occasional mixing by vortex facilitates extrac-
tion and removal of non-fl agellum-associated microtubules in
the cells.
6. The fi nal pellet containing extracted fl agellum and associated
structures [
6
,
7
] was washed three times with PBS before fur-
ther processing for mass spectrometry.
Purifi cation of
T. brucei
fl agellum only, without associated basal
bodies or bilobe, was achieved following a previously published
protocol [
10
] with modifi cations.
3.1.2 Purifi cation
and Extraction
of T. brucei Flagellum
1. 1,000 ml procyclic
T. brucei
cells (
see
Note 3
) are cultivated
with Cunningham's medium supplemented with 15 % heat-
inactivated fetal calf serum at 28 °C in a 2-L spinner bottle
with gentle mixing at ~50 rpm, to near stationary phase
(~1.6 × 10
7
/ml) (
see
Note 4
). The cells are harvested by cen-
trifugation at 2,000 ×
g
for 10 min.
2. Wash the cells twice with 30 ml buffer A.
3. Resuspend the cells in 30 ml buffer A supplemented with 1 %
BSA, 0.1 mM CaCl
2
, and protease inhibitors.
4. Perform 5 sonication cycles, with each cycle containing 20 s
pulse at 20 % of maximum output (Sonics Vibra-Cell, 130 W)
and 40 s rest in between pulses, to detach the fl agella from the
cell bodies (
see
Note 5
).
5. Centrifuge at 800 ×
g
for 10 min to remove unbroken cells.
6. Transfer the supernatant to a 30-ml polycarbonate tube.
Centrifuge at 6,900 ×
g
for 15 min at 4 °C.
7. Remove the supernatant and resuspend the pellet in 4 ml buf-
fer A supplemented with protease inhibitors.
8. Carefully load the resuspended pellet on top of a 6 ml 0.61 M
sucrose cushion. Centrifuge at 1,200 ×
g
for 15 min in a swing-
out rotor (such as SW 41 Ti, Beckman Coulter, Inc.).
