Biology Reference
In-Depth Information
Fig. 1
Isolation of the cytosolic fraction from
Arabidopsis
cell cultures for proteomic analysis. (
a
) Protoplasts
from 7-day-old cell cultures are gently disrupted with a Potter-Elvehjem homogenizer. Broken protoplasts are
subjected to three successive centrifugation steps of: 800×
g
for 15 min, 10,000×
g
for 15 min, and
100,000 ×
g
for 1 h. These remove unbroken cells, large cellular debris, organelles, and small vesicular com-
ponents from the cytosolic fraction (Cytosol). (
b
) After diafi ltration and concentration of cytosolic proteins using
5 kDa cut-off spin columns, the sample is separated by SDS-PAGE. Note the banding pattern of the cytosolic
fraction differs to that of intact protoplasts, the 10,000 ×
g
and 100,000 ×
g
pellet fractions. These gels are also
subjected to immunological analysis to evaluate organelle contamination in the cytosolic fraction (
see
Fig.
2
).
(
c
) Isolated cytosolic proteins are digested with trypsin, desalted and fractioned by strong cation exchange
(SCX) liquid chromatography to create multiple fractions for peptide identifi cation by LC-MS/MS analysis
(offl ine MudPIT). Peptides are detected by a UV detector set at a wavelength of 214 nm and collected in 15-16
fractions, with most peptides eluting from the SCX column within the fi rst half of the 140 min run
3. Isolation of the cytosolic fraction
(a) Centrifuge the homogenate at 800 ×
g
for 15 min at 4 °C
and discard the pellet containing unbroken cells, large
cellular debris, and nuclei (Fig.
1
).
(b) Centrifuge the supernatant at 10,000×
g
for 15 min at
4 °C and discard the pellet, which is enriched in mito-
chondria and plastids (Fig.
1
).
(c) Centrifuge the supernatant at 100,000 ×
g
for 1 h at 4 °C
and collect the supernatant (cytosolic fraction). The dis-
carded pellet contains secretory pathway components,
plasma membrane, Golgi, and ER (
see
Note 9
and Fig.
1
).
