Biology Reference
In-Depth Information
Broken plasd
40% (v/v) Percoll
intact plasd
80% (v/v) Percoll
Fig.
1
Isolation of intact plastid from rice seedling
6. Suspend the pellet with an appropriate volume of plastid
dilution buffer. Check the chloroplast integrity/quality under
a light microscope (
see
Note 18
). The suspension can be pre-
served at −80 °C (
see
Note 19
). For proteomics application,
dissolve the pellet in fi ve times the volume of protein extrac-
tion buffer.
3.4 Protein
Extraction
The method described here is for quantitative proteomic analysis.
1. Suspend the samples in protein extraction buffer and sonicate.
2. Centrifuge the homogenate at 12,000 ×
g
for 5 min at 4 °C and
transfer the supernatant to a new tube.
3. Add 1/10 volume of 100 % trichloroacetic acid (TCA) to the
supernatant and mix. Incubate the solution on ice for 15 min
to precipitate proteins.
4. Centrifuge the homogenate at 10,000 ×
g
for 5 min at 4 °C and
discard the supernatant.
5. Add 1 mL of ice-cold acetone to the pellet and mix. Centrifuge
the homogenate at 10,000 ×
g
for 10 min at 4 °C. Discard the
supernatant.
6. Repeat
step 5
three to fi ve times (
see
Note 20
).
7. After drying the pellet, resuspend it in 20
μ
L of dissolution
L of denaturant (2 % SDS). Both buffers are
supplied in the iTRAQ Reagents kit (
see
Note 21
).
8. Measure the protein concentration. 5-50
buffer and 1
μ
μ
g of protein is
desirable for iTRAQ labeling.
3.5
iTRAQ Labeling
The peptide labeling with iTRAQ is in accordance with the
manufacturer's protocol (
http://www.absciex.com/downloads/
mass-spectrometry-literature
)
. The strategy of quantitative
proteomic analysis using iTRAQ is summarized in Fig.
2
.
