Biology Reference
In-Depth Information
15. Lysis solution: 30 % sucrose (w/v), 2 % SDS (w/v), 5 %
β
-mercaptoethanol (v/v).
16. Lysis-saturated phenol (1:1 ratio) solution: mix 1 volume of
10 mM Tris-HCl, pH 8.0 buffer-saturated phenol (Sigma
®
)
with 1 volume of lysis solution.
17. Solubilization solution: 7 M urea, 2 M thiourea, 4 % (w/v)
3[(3-Cholamidopropyl)-dimethylammonium]-
propanesulfonic acid (CHAPS), 0.5 % (w/v) Triton X-100
and 20 mM DTT.
18. Bovine serum albumin (BSA) solution (1 mg/mL).
19. IPG strip rehydration solution: 7 M urea, 2 M thiourea, 4 %
(w/v) CHAPS, 0.01 % (w/v) bromophenol blue, 100 mM
DTT, and 0.2 % (v/v) of 3-10 ampholytes (Bio-Rad
®
).
20. IPG strip equilibrium solution: 375 mM Tris-HCl, pH 8.8,
6 M urea, 20 % (v/v) glycerol, 2 % (w/v) SDS.
21. 10 % (w/v) sodium dodecyl sulfate (SDS) solution.
22. 10 % (w/v) ammonium persulfate (APS) solution.
23. Electrophoresis stacking gel buffer: 1.5 M Tris-HCl, pH 8.8.
24. Electrophoresis running buffer: 50 mM Tris-HCl, pH 8,
192 mM Glycine, 1 % (w/v) SDS.
25. Electrophoresis sample buffer: 0.5 M Tris-HCl, pH 6.8, 10 %
(v/v) glycerol, 10 % (w/v) SDS, 0.5 % (w/v) bromophenol blue,
5 % (v/v)
26. Sealing solution: 0.5 % (w/v) agarose and 0.01 % (w/v) bro-
mophenol blue.
27. Gel staining Coomassie colloidal suspension: 0.06 M
(NH
4
)
2
SO
4
, 20 % (v/v) methanol, 1.9 % (w/w) H
3
PO
4
, 0,1 %
(w/v) Coomassie G-250.
28. Gel destaining solution A: 0.1 M Tris-H
3
PO
4
, pH 6.5.
29. Gel destaining solution B: 25 % (v/v) methanol.
30. Gel destaining solution C: 20 % (w/v) ammonium sulfate.
31. Gel fi xing solution: 10 % (v/v) methanol, 7 % (w/v) acetic acid.
32. Gel washing solution: 20 mM ammonium bicarbonate, 50 %
(v/v) acetonitrile.
β
-mercaptoethanol.
3
Methods
The workfl ow in Fig.
1
is stated in order to choose the conditions
to evaluate proteins associated to carnation defense against the
pathogen responsible for vascular wilting by 2-DE. This workfl ow
may be applied to other plant species in similar studies.
