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Et al. (2003) Diversity of puroindolines as revealed by two-dimensional electrophoresis. Proteomics 3,168–174 22. , and Branlard, G. (2002) Proteomic analysis of amphiphilic proteins of hexaploid wheat. Proteomics 2, 632–641. 23. C. J. (1996) An improved 1-D SDS-PAGE method for the identification of three bread wheat “waxy” proteins. J. Cereal Sci. 23, 191–193. 24. , and Branlard, G. (2000) Allelic variants of granule-bound starch synthase proteins in European bread wheat varieties. Plant Breeding 119, 305–309.

14. Weigh the centrifuge tube containing the dry pellet. 2. Protein Solubilization 1. Resuspend the powder in the solubilization buffer. We use between 10 and 30 µL of buffer per mg of powder. This buffer contains a mixture of chaotropes (urea, thiourea), detergents (Triton-X 100, CHAPS), reductants (DTT), and carrier ampholytes (IPG buffer). 2. , cellular fragments). 3. Pour the supernatant into a clean tube. 4. Centrifuge at 400g for 4 min at room temperature, and pour the supernatant into a clean tube.

Diseased plants, such as those infected with the vascular wilt fungus Fusarium oxysporum, have much lower sap yields. 3. Protein or sugar concentration can be used as a measure for phloem contamination; low concentrations of both substances indicate a high purity of xylem sap samples. 4. Poly- and oligosaccharides react with common reagents to measure protein concentration, like Bradford and bicinchoninic acid (BCA; Sigma). Their presence in xylem sap leads to an overestimation of protein content.

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