Biology Reference
In-Depth Information
2.2 Reagents
and Solutions
Prepare all solutions in distilled water (conductivity lower than
3
S·cm
-1
). Store all solutions at 4 °C (unless otherwise indicated).
1. Extraction solution for albumins (ES1): 10 mM Tris-HCl,
pH 7.5, 0.1 % (w/v) DTT, 10 mM MgCl2, 10 mM CaCl2.
2. Extraction solution for globulins (ES2): 10 mM Tris-HCl,
pH 7.5, 1 M NaCl, 0.1 % (w/v) DTT, 10 mM EDTA, 10 mM
EGTA.
3. Extraction solution for prolamins (ES3): 75 % (v/v) ethanol.
4. Extraction solution for glutelins (ES4): 50 mM sodium borate
buffer, pH 10, 2 % (w/v) DTT, 1 % (w/v) SDS. Store this
solution at room temperature.
5. Solubilization solution: 7 M urea, 2 M thiourea, 4 % (w/v)
CHAPS, 2 % (v/v) Tritón-X-100, 100 mM DTT. Add DTT to
10 mM just before use.
6. Bradford reagent (Sigma-Aldrich).
7. SDS-PAGE running buffer: 25 mM Tris-HCl, pH 8, 192 mM
glycine, 0.1 % (w/v) SDS.
8. Acrylamide/Bis-acrylamide solution (30 %) commercial solu-
tion (Bio-Rad).
9. Ammonium persulfate: 10 % (w/v).
10. SDS: 10 % (w/v).
11. Resolving gel buffer: 1.5 M Tris-HCl, pH 8.8.
12. Stacking gel buffer: 0.5 M Tris-HCl pH 6.8.
13. Colloidal Coomassie staining gel: 10 % (w/v) ammonium sul-
fate, 3 % (w/v) phosphoric acid, 0.1 % (w/v) Coomassie
Brilliant Blue (CBB) G250, 20 % (v/v) methanol.
14. Tris-phosphoric solution: 0.1 M Tris-phosphoric acid, pH 6.5.
15. Ammonium sulfate: 20 % (w/v).
16. Methanol: 25 % (v/v).
17. TEMED, commercial product (Sigma) (
see
Note 2
).
μ
3
Methods
Holm oak mature acorns (with brown pericarp) were used in our
research. Once harvested, acorns were selected, cleaned and stored
in airtight polyethylene bags at 4 ± 1 °C. Acorns were scarifi ed with
a knife by making transversal and longitudinal cuts, thus permit-
ting the pericarp to be rapidly removed. Once peeled off, cotyle-
dons (including embryo axes) were triturated in an analytical mill
to obtain a homogeneous fi ne fl our (Fig.
1
). The obtained powder
was weighed and stored in a desiccator at 4 ± 1 °C, until protein
extraction [
18
].
3.1 Plant Material
