Biology Reference
In-Depth Information
4. Fractions were collected at a pH interval of 0.3 in a 96 deep-
well plate in an FC/I module (fraction collector/injection).
At 170 min, the column was washed with 1 M NaCl for
15 min (
see
Note 5
).
5. Finally, the fi rst-dimension column was washed with water for
45 min.
6. Proteins were then separated in the second-dimension high-
performance reversed-phase (HPRP). When the fi rst-
dimension separation was completed (170 min), the pI
fractions from the fi rst dimension were then automatically run
on the second dimension as indicated in the ProteomeLab
PF 2D method: Column, HPRP 2D Column. Mobile phase,
solvent A and B. Flow rate, 0.75 ml/min. Temperature, 50 °C.
Detection, UV 214 nm and back pressure, 1,500-2,000 psi.
7. Prime lines of the HPRP module with solvent A and solvent B.
8. Injection of 200
μ
l of sample for each fi rst-dimension
fraction.
9. After the sample has been applied to the column from the
FC/I module, a gradient of 0-100 % solvent B for 30 min,
fl ow rate 0.75 ml/min, is run.
10. Run 100 % solvent B for 5 min followed by 100 % solvent A
for 10 min at a fl ow rate of 0.75 ml/min before the next
sample.
11. The method automatically saves the raw UV absorbance data
for each second-dimension analysis of the chromatofocusing
fractions for protein mapping and data analysis using 32Karat
and ProteoVUE™ in the PF 2D software Suite.
12. In Fig.
2
the chromatofocusing using the ProteomeLab PF
2D system was presented. The fraction of proteins resolved by
the ProteomeLab PF 2D gradient (pH 8.0-4.0) corresponded
to 0.625 mg. This represented 25 % of the 2.5 mg of total
proteins that were initially injected in the column. About
1.1 mg, 44 % of the total proteins were resolved before the pH
gradient, thus representing proteins that were not retained by
the column. After the pH gradient, the column was washed
with a high salinity buffer and different peaks were observed
corresponding to 0.775 mg, about 31 % of the total proteins
(
see
Notes 6
and
7
).
In summary we can conclude that both methodologies, 2DE and
liquid chromatography, are complementary methods to analyze
complex protein extracts and they can be used in parallel to acquire
a wider perspective and a better understanding, in this case, of the
embryo proteome. Therefore, the choice of the methodology to
be applied will depend on the proteins of interest (Fig.
3
).
3.5 Combination
of 2DE and LC
for Plant Proteomics
Analysis
