Biology Reference
In-Depth Information
2.6 Bioinformatic
Analysis of Proteins
Identifi ed by MS
1. TargetP:
http://www.cbs.dtu.dk/services/TargetP/
[
23
].
2. Predotar:
http://urgi.versailles.inra.fr/predotar/predotar.
html
[
24
].
3. PSORT:
http://psort.hgc.jp/form.html
.
4. GPIsom:
http://gpi.unibe.ch/
[
25
].
5. BigPI:
http://mendel.imp.ac.at/sat/gpi/gpi_server.html
[
26
].
6. PROSITE:
http://prosite.expasy.org/
[
27
].
7. PFAM:
http://pfam.sanger.ac.uk/
[
28
].
8. InterProScan:
http://www.ebi.ac.uk/Tools/pfa/iprscan/
[
29
].
9. BLAST:
http://blast.ncbi.nlm.nih.gov/Blast.cgi
[
30
].
10. CD-search:
http://www.ncbi.nlm.nih.gov/Structure/cdd/
wrpsb.cgi
[
31
].
11. ProTerNyc:
http://www.polebio.lrsv.ups-tlse.fr/ProTerNyc/
[
32
].
3
Methods
The overall strategy from xylem sap harvesting to protein identifi -
cation by MS and bioinformatics analysis is schematized on Fig.
1
.
3.1 Plant Production
1. Sow seeds on loam. Place pots in the greenhouse. After 15
days, repot seedlings individually in peat pots containing loam.
Place pots in the greenhouse for 3-4 weeks.
2. Water plants regularly (
see
Note 7
). Give fertilizer once a week.
3.2 Xylem Sap
Harvesting
( See Note 8
)
1. Put 5-6 week-old plants (3-4 pairs of leaves) in mini-
greenhouses placed in the lab at room temperature (
see
Notes
9
-
10
) for one night. Pots must be saturated with water.
2. Cut stems horizontally above the fi rst pair of leaves with a
razor blade (
see
Note 11
). Rinse thoroughly with water and
dry with paper towels.
3. Collect regularly drops of xylem sap exuding on the surface of
the cut with a Micropipette (Fig.
1
) (
see
Note 12
). Store sap at
−20 °C if rapidly used or at −80 °C for long conservation.
4. Harvest during 10-12 h. Number each fraction. Discard the
two fi rst fractions and pool other fractions (
see
Note 13
).
Routinely, 0.7 mL/plant/day of xylem sap was collected.
3.3 Preparation
of the Protein Extract
1. After harvesting of the xylem sap, dialyze the samples (about
20 mL) against buffer B1 in a Mega GeBAfl ex-tube. Dialysis is
performed against a large volume of buffer B1 (100-1,000-
fold that of the sample) for 3 h at 4 °C. Change dialysis buffer
B1 and continue dialysis for another 3 h.
