Biology Reference
In-Depth Information
2.1
Plant Production
1.
Brassica oleracea
var Bartolo F1 calibrated seeds treated with a
fungicide (Bejo, France).
2. Universal compost for seedlings and cuttings, containing peat
moss, sphagnum peat brown, clay, and perlite (catalog number
A16805, AGRI Garonne, Castelginest, France).
3. Peat pots composed of 80 % wood fi ber and 20 % peat (catalog
number 53803, AGRI Garonne).
4. Universal fertilizer (Universel ALGOFLASH NPK 6/6/6).
5. Greenhouse 22-27 °C, 16 h of daylight, 10,000 lx, 55-75 % of
relative humidity.
1. Mini-greenhouses (Stewart) 52 × 42 × 24 cm.
2. Rasor blades.
2.2 Xylem Sap
Harvesting
1. Dialysis tubes: Mega GeBAfl ex-tubes MWCO 12-14 kDa
(catalog number D080-6/10-10, Gene Bio-Application
L.T.D., Yavne, Israel) (
see
Note 1
).
2. 1 M Tris-HCl pH 7.4 stock solution. Weigh 12.1 g of Tris
(catalog number 26.128-3094-B, EUROMEDEX,
Mundolsheim, France). Add water to a volume of 90 mL. Mix
and adjust pH with HCl. Make up to 100 mL with water.
Store at 4 °C.
3. 5 M NaCl stock solution. Weigh 29.2 g of NaCl. Add water to
a volume of 100 mL. Store at 4 °C.
4. 10 mM MgCl
2
/MnCl
2
/CaCl
2
stock solution. Weigh 0.244 g
MgCl
2
hexahydrate (catalog number M9272, Sigma-Aldrich,
Saint-Louis, MO), 0.237 g MnCl
2
tetrahydrate (catalog num-
ber 221279, Sigma-Aldrich) and 0.176 g CaCl
2
dihydrate (cat-
alog number 223506, Sigma-Aldrich) (
see
Note 2
). Add water
to a volume of 250 mL. Store at 4 °C.
5. Dialysis buffer B1: 20 mM Tris-HCl pH 7.4, 150 mM NaCl,
1 mM MgCl
2
/MnCl
2
/CaCl
2
. Mix 20 mL of 1 M Tris-HCl
pH 7.4, 30 mL of 5 M NaCl, 100 mL of 10 mM MgCl
2
/
MnCl
2
/CaCl
2
stock solutions. Add water to a volume of 1 L
(
see
Note 3
). Store at 4 °C.
6. Desalting columns: Econo-Pac 10 DG columns (catalog num-
ber 732-2010, Bio-Rad, Hercules, CA)
7. Desalting buffer B2: 0.1 M ammonium formate. Weigh 6.3 g
of ammonium formate (catalog number 156264, Sigma-
Aldrich). Add water to a volume of 1 L. Store at 4 °C.
8. Bradford method reagent: Coomassie Protein assay Kit (catalog
number UPF8640, Uptima, Interchim, Montluçon, France).
9. Lyophilizator Christ Alpha 1-4 LSC (Fischer Scientifi c,
Illkirch, France).
2.3 Preparation
of the Protein Extract
