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followed by mass spectrometry (MS) for their identifi cation [ 3 ].
The heterogeneity of polypeptide molecular size, charge, hydro-
phobicity, complexity, and cellular distribution makes it almost
impossible to capture and solubilize the entire complement of pro-
teins in a given sample [ 4 , 5 ].
More recently, proteome analyses have been performed in a
“gel-less” condition by using protein fractionation procedures
based entirely on liquid chromatography (LC). Here we use a
system of two-dimensional liquid chromatography based on a
high-performance chromatofocusing in the first dimension,
followed by high-resolution reversed-phase chromatography in
the second dimension [ 6 - 8 ].
In the present work we have used the conventional 2-D gel
electrophoresis and liquid chromatography. We have isolated wheat
embryos from the Tunisian cultivar and analyzed their total pro-
tein extracts in a different pH range with two different separation
techniques [ 1 ].
2
Materials
Triticum durum Desf., salt- and drought-tolerant Tunisian cultivar
(Oum Rabiaa) of wheat.
2.1 Plant Material
2.2 Equipment
The materials 1-11 were all purchased from GE Healthcare Life
Sciences.
1. Isoelectrofocusing (IEF), IPGphor II Isoelectric Focusing
Unit.
2. IPGphor Strip Holders.
3. Second-dimension Ettan DALT six systems.
4. SDS gel-casting box.
5. Cassette rack.
6. Glass plates.
7. Thermostatic circulator (Multitemp III).
8. Power supply (Multidrive XL).
9. Laboratory shaker.
10. ImageScanner.
11. ImageMaster 2D Platinum 5.0 software.
12. LC, ProteomeLab PF-2D (Beckman Coulter).
13. LC software, 32 Karat, and ProteoVUE™ (Beckman Coulter).
2.3 Products
and Stock Solutions
1. Lysis buffer 1: 7 M urea, 2 M thiourea, 4 % CHAPS, 50 mM
Tris-HCl, pH 8.0, supplemented with 53 u/mL DNase I,
4.9 u/mL RNase, a cocktail of protease inhibitors (1 mM
PMSF, 0.1 mM pepstatin, 2 mM leupeptine 1 mM E-64, and
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