Low molecular weight seed proteins are restricted to the embryo
tải về 109.87 Kb. Chế độ xem pdf
|
| 3. Conclusion
Buckwheat (F. esculentum) seeds can be separated into husk, embryo and endosperm. No low M.W. proteins, which are known to cause allergy, could be detected on electrophero- grams of buckwheat endosperm. There appeared tissue spe- cific proteins of different size classes of the endosperm and embryo tissues. It seems that endosperm cells because of their high specialization contain very low amount of low M.W. pro- teins, including small housekeeping proteins. With improved technology it should be possible to separate endosperm from embryo on the larger scale, which would enable products to be made from buckwheat flour that could be more appropri- ate for patients with hypersensitivity to buckwheat flour than buckwheat flour products generally used. 4. Methods 4.1. Plant material Common buckwheat (F. esculentum Moench) cv. Siva, was field-grown in the experimental garden of the Biotechnical faculty, University of Ljubljana. Prior to dissection, the unhusked buckwheat seeds were soaked for 1 h in distilled water. The husk was removed from each seed under a magnifying glass for analysis; the en- dosperm and embryo (cotyledons) were carefully separated and placed into 1.5 ml centrifuge tubes. Each endosperm and embryo was smashed separately with a glass rod in 300 µl of extraction buffer (0.5 M Tris (hy- droxymethyl) aminomethane hydrochloride (Tris–HCl) (pH 6.8), 20% glycerol, 4% SDS, 3% 2-mercaptoethanol, bro- mophenol blue). After 2 h of extraction, the samples were incubated in a water bath at 95 °C for 5 min, centrifuged for 15 min at 10,000 rpm and 15 µl of supernatant was applied for electrophoresis. 4.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis) Separation gels (15% acrylamide) (1.5 M Tris–HCl (pH 8.8), 15% acrylamide stock, 1% SDS, 1% ammonium persul- fate, tetramethylethylenediamine (TEMED)) and concentra- tion gels (1.5 M Tris–HCl (pH 6.8), 4% acrylamide stock, 1% SDS, 1% ammonium persulfate, TEMED) were prepared as described [40] . Vertical electrophoresis (Hoefer, San Fran- cisco) was carried out at 40 mA for 1 h, and about 3 h at 60 mA. Proteins were stained for 24 h with 0.05% Coo- massie Brilliant Blue R-250, 5% ethanol, and 12% TCA, and destained in deionized water. For standards we used wide range molecular markers SigmaMarker™ M 4038 (M.W. 6500–205,000). tải về 109.87 Kb. Chia sẻ với bạn bè của bạn:
|