Biology Reference
In-Depth Information
research objectives [
13
]. In our experience, protein extraction
using the TCA/acetone and TCA/acetone-phenol protocols pro-
vided the best results with a large variety of plant species [
13
-
17
].
Protein solubilization is a critical step. Detergents as CHAPS and
chaotropic agents such as urea and thiourea must be used in the
solubilization solutions to help in the hydrophobic protein solubi-
lization, obtaining a higher protein yield [
9
,
16
-
19
]. Protein sepa-
ration with SDS-PAGE is a quick and accurate technique for plant
proteins, especially in the case of comparative proteomics with
large numbers of samples to be analyzed. Using appropriate soft-
ware, SDS-PAGE is a simple, reliable technique for fi nger-printing
crude extracts, and it is especially useful in the case of hydrophobic
and low molecular-weight proteins [
20
]. Furthermore, SDS-PAGE
is a good approach to obtain preliminary results before performing
2-DE analysis [
15
,
16
]. Two-DE has been used for separating and
displaying the components of large protein complexes, and it has
been a reliable tool to study natural variability in several plants spe-
cies [
1
,
3
-
5
,
21
,
22
]. After staining (we currently use colloidal
Coomassie staining [
23
]), images are digitized and analyzed with
appropriate software [
24
]. Quantitative proteomics data are classi-
cally assessed by univariate statistics (
t
-test, Mann-Whitney,
ANOVA, Kruskal-Wallis), but these methods increase the possibil-
ity of false positives, are negatively affected by the raw structure of
proteomics data, and they cannot detect trends and protein rela-
tions [
25
-
28
]. On the other hand, the analysis employing multi-
variate approaches (i.e., Principal Components, Self Organizing
Maps) are described to be more effective, because of its capacity to
reduce the complexity of the data, predict trends and also for being
less affected by data structure [
26
-
28
]. Furthermore, data analysis
can be used to discriminate and establish phylogenetic relation-
ships among populations and genotyping, as well as to correlate
the profi le with edaphoclimatic characteristics and morphometric
parameters. Finally, major differential bands or spots are excised
from gels and subjected, after tryptic digestion, to MS analysis and
their identifi cation [
29
,
30
].
2
Materials
Mention of specifi c companies or pieces of equipment is not man-
datory, and it does not represent an endorsement by the authors.
All the chemicals should be of analytical grade.
1. Liquid Nitrogen.
2. Trichloroacetic acid (TCA) (10 % w/v)/acetone (80 % v/v)
solution. Store at −20 °C and use directly from the freezer.
3. 0.1 M Ammonium acetate/methanol (100 % and 80 % v/v)
solution. Store at −20 °C and use directly from the freezer.
2.1 Reagents,
Solutions and Buffers
2.1.1
Protein Extraction
