Biology Reference
In-Depth Information
6. Additional assays must be done for isoelectric point assigna-
tion when the best separation is achieved with the nonlinear
gradient. Add 5
L of the commercial markers (Bio-Rad
®
Specialty Standard IEF) (
see
Note 21
), and complete to
400
μ
L using IPG strip rehydration solution. Shake vigorously
by vortexing.
7. Pour gently the previously prepared mixture creating a line in
one of the rails of the IPG strip focusing tray, avoiding the
formation of bubbles. Repeat the procedure previously
described from
step 3
to
7
in Subheading
3.6.1
for rehydra-
tion and isoelectrofocusing.
8. Once the isoelectric focusing is fi nished, remove the strip from
the system and keep it vertically to dispose most of the mineral
oil. Add coomassie colloidal solution to the IPG strip directly
according to numeral 3-6 in Subheading
3.5.2
.
9. Measure using a rule, the position from the left end mark
(pH 3) to each band separated on the previously stained strip.
Assign the isoelectric point for each band according to this
measured position and the marker composition.
10. Compare the position of these bands and the position of most
intense spots in a 2-D gel obtained using the same type of IPG
strip NL. Assign isoelectric points for these representative
spots according to its displacement from the pH 3 end mark
on the gel and the position of the bands in the strip.
11. Assign the pI for the most intense and representative spots
according to the described comparison during the PDQuest
Analysis. The assignment of pI for the other proteins is made
automatically.
μ
The extraction and separation (1-DE and 2-DE) conditions
described above were required to carry out the proteome analysis
in carnation stems and roots during the infection with
Fod
, using
gel-based proteomic techniques, as different authors have previ-
ously described in other plant-pathogen interactions [
4
,
5
,
13
,
14
].
3.7 Downstream
Steps in the Workfl ow
and Main Results
MS analysis and protein identifi cation is currently carried out at the
SCAI (Servicio Central de Apoyo a la Investigación, Universidad
de Cordoba).
1. Spots are cut from the gel using a station ProPic Investigator
(GenomicSolution).
2. The resulting gel fragments are subjected to digestion with tryp-
sin and the resulting peptides analyzed by MALDI-TOF/TOF.
3. The identifi cation is based on comparing the MS fragmenta-
tion patterns and corresponding MS/MS, on the NCBInr
database using as search engine MASCOT (MatrixScience),
and limiting the taxonomic category to plants (
Viridiplantae
).
