Laurière M, Bouchez I, Doyen C, Eynard L
Laboratoire de Chimie Biologique, Centre INRA de Grignon, Thiverval-Grignon, France.
Electrophoresis. 1996 Mar;17(3):497-501. doi: 10.1002/elps.1150170312.
Two-dimensional electrophoresis with acid-polyacrylamide gel electrophoresis (PAGE), followed by sodium dodecyl sulfate (SDS)-PAGE and SDS-PAGE of unreduced polypeptides followed by SDS-PAGE under reducing conditions, were used to separate and identify the different subgroups of gliadins and glutenins and to distinguish between covalent and noncovalent polymers of glutenins. Gels were blotted under semidry conditions according to Laurière (Anal. Biochem. 1993, 212, 206-211) to allow large polymers of glutenins to be transferred efficiently. Glycosylated polypeptides were detected on blots using either the method of Haselbeck and Hösel (Glycoconjugate J. 1990, 7, 63-74), or using anti-(xylose-containing N-glycan) antibodies (Laurière et al., Plant Physiol 1989, 90, 1182-1188). High and low molecular weight glutenin subunits were shown to aggregate through both disulfide bridges and noncovalent protein-to-protein interactions. Aggregated gamma-gliadins were also demonstrated. Glycans were detected on both gliadin and glutenin polypeptides. Covalently aggregated low molecular weight glutenins were shown to contain N-glycans with xylose, which demonstrated their sorting in the Golgi apparatus.
采用酸性聚丙烯酰胺凝胶电泳（PAGE）进行二维电泳，随后进行十二烷基硫酸钠（SDS）-PAGE，以及对未还原的多肽进行SDS-PAGE，然后在还原条件下进行SDS-PAGE，用于分离和鉴定醇溶蛋白和谷蛋白的不同亚组，并区分谷蛋白的共价和非共价聚合物。根据Laurière（《分析生物化学》，1993年，212卷，206 - 211页）的方法，在半干条件下对凝胶进行印迹，以使谷蛋白的大聚合物能够高效转移。使用Haselbeck和Hösel的方法（《糖缀合物杂志》，1990年，7卷，63 - 74页）或使用抗（含木糖的N-聚糖）抗体（Laurière等人，《植物生理学》，1989年，90卷，1182 - 1188页）在印迹上检测糖基化多肽。结果表明，高分子量和低分子量谷蛋白亚基通过二硫键和非共价蛋白质-蛋白质相互作用聚集。还证实了聚集的γ-醇溶蛋白。在醇溶蛋白和谷蛋白多肽上均检测到聚糖。共价聚集的低分子量谷蛋白被证明含有带木糖的N-聚糖，这表明它们在高尔基体中的分选情况。
