Biomedical Engineering Reference
In-Depth Information
9. Collect the top and the interface layers (~4.5 ml), mix well and
load on top of another 0.61 M sucrose cushion, and repeat
step 8
. Transfer the top and the interface layers (~5 ml) into a
15-ml Falcon tube.
10. Set up the sucrose step gradient in two ultraclear SW 41 Ti
centrifuge tubes as follows:
Place the tubes in a steady tube rack and avoid any distur-
bance throughout the procedure. Add 2 ml of 2 M sucrose
solution at the bottom of each centrifuge tube. Gently overlay
2 ml from each of 1.84, 1.66, and 1.5 M sucrose solutions,
respectively. To avoid mixing the different solutions, hold the
pipette tip just above the meniscus of the solution in the tube
and pipette the solutions by letting them run down the tube
wall. Carefully load the material from
step 9
, ~2.5 ml to the
top of each tube. Balance the two tubes to exact same weight
(
see
Note 6
).
11. Centrifuge overnight (16-20 h) at 4 °C in a SW 41 Ti rotor at
75,000 ×
g
(28,000 rpm) (
see
Note 7
).
12. Place the centrifuge tubes containing the sucrose gradient on a
tube rack on ice. Flagella are enriched at the 1.66/1.84 M
interface. Carefully remove all the top layers using a pipette to
reach the 1.66 M layer.
13. Collect the 1.66/1.84 M interface. Pool the fractions together
from the two tubes (~1 ml) and dilute 10× with buffer
A. Centrifuge at 75,000 ×
g
in a SW 41 Ti rotor for 30 min to
concentrate the fl agella.
14. Extract the purifi ed fl agella as described above in “Purifi cation
of
T. brucei
Flagellar Complex,” following
steps 3
-
6
.
Solubilize the fl agellum and fl agellar complex samples prepared
above in a small amount of dissolution buffer (
see
Note 8
). Total
protein concentration can be determined using a spectrophotometry-
based protein assay (e.g., BCA assay, Pierce) or other appropriate
quantifi cation methods. Adjust total protein concentration to
5
3.1.3 Comparative
Proteomics by Isobaric Tag
for Relative and Absolute
Quantitation (iTRAQ)
l using 0.5 M TEAB. Further processing of the samples
follows the iTRAQ protocols from Applied Biosystems and
Sadowski et al. [
11
].
μ
g/
μ
1. To each tube containing 20
g of pro-
tein each tube, two tubes of fl agellum complex, and two tubes
of fl agellum samples), add 2
μ
l protein sample (100
μ
μ
l reducing reagent. Mix well and
incubate at 60 °C for 1 h.
2. To each tube, add 1
l of cysteine blocking reagent (
see
Note 9
),
mix well, and incubate at room temperature for 10 min.
3. Dilute each sample using 50 mM TEAB to reduce SDS
concentration to 0.05 % or less.
μ
