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以硝酸银诱导的大豆子叶为材料,初步探讨了二维电泳技术在分离大豆子叶微粒体膜结合蛋白中的应用,研究了重新水化溶液的组成、加样量、等电聚焦的total voltage hours以及不同pH梯度范围IPG(固相pH梯度)胶条等因素对膜蛋白的溶解和二维电泳图像的影响。结果表明对于70×3×0.5mmIPG胶条,能够溶解较多蛋白质和获得高质量二维电泳图像的优化条件为:由尿素、硫脲、三丁基膦和C7BZO组成的重新水化溶液(TUC7T2)、80μg的加样量和20000vhr的totalvoltage hour。在此优化条件下,与宽范围pH梯度IPG胶条相比,使用窄范围pH梯度胶条可以观察到更多的和质量更好的蛋白质点,结果发现至少有13个点只存在于硝酸银诱导的样品中,而不存在于对照中,其代表的蛋白质的等电点在4.0-8.0。
Using silver nitrate-induced soybean cotyledon as material, the application of two-dimensional electrophoresis in the isolation of microsomal membrane-bound protein from soybean cotyledons was discussed. The composition of the rehydration solution, the amount of sample, the total voltage of isoelectric focusing And different pH gradient range IPG (solid pH gradient) strips and other factors on the dissolution of membrane proteins and two-dimensional electrophoresis images. The results showed that for the 70 × 3 × 0.5mmIPG strips, the optimum conditions for dissolving more proteins and obtaining high-quality two-dimensional electrophoresis images are: rehydration solution consisting of urea, thiourea, tributylphosphine and C7BZO (TUC7T2 ), A loading of 80 μg and a totalvoltage hour of 20000 vhr. Under this optimized condition, more and better quality protein spots were observed with the narrow range pH gradient strips compared to the wide range of pH gradient IPG strips and found that at least 13 spots were only present in the silver nitrate Induced sample, but not in the control, represented a protein with an isoelectric point of 4.0-8.0.