Sucrose → Tris Tricine

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Tris tricine SDS-PAGE electrophoresis was performed according to Schagger and von Sagow [1987] using precast 16.5% T gels (Biorad, Hercules, Calif.). The anode buffer consisted of 0.2M Tris-HCl, pH 8.9 and the cathode buffer consisted of 0.1M Tris-HCl, 0.1M Tricine, 0.1% SDS, pH 8.25. Samples were diluted in 10 μl of 50 mM Tris-HCl, 4% (w/v) SDS 12% (w/v) sucrose, 5% (v/v) β morcaptoethanol, and trace of bromophenol blue, pH 6.8. After denaturation at 95° C. for 5 min, samples were loaded onto the gel. Gels were run at 80V for 3 hours. After electrophoresis, gels were fixed in 40% methanol, 10% acetic acid for 30 min. Gels were then stained with Colloidal blue coomassie G250 overnight and destained in 30% methanol. Bands to be identified were immediately cut, placed in an eppendorf and kept at 4° C. until further analysis. The apparent molecular masses were determined by running polypeptide molecular weight (MW) standards: Triosephophate isomerase MW 26,625; Myoglobin MW 16,950; α-lactalbumin MW 14,437; Aprotinin MW 6,512; Insulin b chain, oxidised MW 3,496 and Bacitracin MW 1,423 (Biorad).

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