Biology Reference
In-Depth Information
9. Select a good image as a reference with a clear and representa-
tive spot pattern and with a minimum distortion, and align
each of the images to the chosen reference. Select between
three prominent spots to manually assignment, and use the
automatic vector tool to add additional vectors.
10. After automatic spot detection and matching, check manually
the spots with edition tools for correct detection.
11. Set gel groups according to the experimental design and nor-
malize spot volume intensity ratios for each spot.
12. List all the spots together with their normalized volume.
Prior to statistical and phylogenetic analyses, the volume of pixels
for each band or spot is normalized according to the total volume
of bands detected (SDS-PAGE) or to the total volume of valid
spots in each gel (2-DE), respectively. Then, they are log-
transformed, following the recommendations described by Valledor
and Jorrin [
34
].
A multivariate analysis is carried out on two steps: fi rstly, hier-
archical clustering is performed to check the entire dataset, and the
results are indicated in dendrograms using the cluster function of
the software used; secondly, the dataset is analyzed by the use of
Principal Component Analysis (PCA). The settings used for the
PCA analysis are: co-variance matrix type, three principal compo-
nents, onefold change, and 0.4 correlation threshold for clusters.
PCA results are shown as a biplot.
Since the employed statistical methods tend to classify popula-
tion together, this information is necessary for studying the possi-
ble correlation between distances and geographical and climate
parameters.
3.6 Phylogenetic and
Statistical Analyses
Spots are manually excised with a scalpel. Protein identifi cation was
carried out according to the Proteomics Service protocols of the
University of Córdoba. Gel plugs are digested with modifi ed por-
cine trypsin (Sequencing grade; Promega), by using an automatic
ProGest digestion station (Genomics Solution). The conditions
are two detained steps for 30 min with 200 mM ammonium bicar-
bonate in 40 % (v/v) ACN at 37 °C; twice washed with 25 mM
ammonium bicarbonate for 5 min and 25 mM ammonium bicar-
bonate in 50 % (v/v) ACN for 15 min respectively; dehydration
with 100 % (v/v) ACN for 5 min and sample dried; hydration
using 10
3.7 Protein
Identifi cation
L trypsin in a solution of 25 mM ammonium bicarbon-
ate at a fi nal concentration of 12.5 ng/
μ
L for 10 min a room tem-
perature, and the digestion is proceeded at 37 °C for 12 h.
Subsequently, digestion is stopped by adding 10
μ
μ
L of a solution
of 0.5 % TFA in water.
