二维电泳技术在大豆子叶微粒体膜结合蛋白研究中的应用

TWO-DIMENSIONAL ELECTROPHORESIS OF MICROSOMAL MEMBRANE BOUND PROTEINS IN SOYBEAN COTYLEDONS

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。

Soybean cotyledons induced by silver nitrate were used to investigate the work of twodimensional electrophoresis （2DE） on the separation of microsomal membrane bound proteins. The following influencing factors on the solubilization of membrane bound proteins and 2DE images were discussed, such as the composing of rehydration solution, sample amount, focusing to- tal voltage hours and different range of immobilized pH gradients （IPG） strips. It was shown that the samples rehydrated in solution making of urea, thiourea, tributyl phosphine and （3-（4-hepty） phenyl 3-hydroxy propyl）（dimethylammonio propane sulfonate）（Ca BZO） could be seen much better 2DE images than that of the other tested rehydration solutions. At the same time, the other running conditions for 70 × 3 × 0.5mm IPG strips were optimized as that the sample amount was 80μg and the focusing total voltage hour was around 20000. Much more clear spots were receivedby the usage of narrow-range spots were found only in the points （pI） were 4.0~8.0. IPG strips than that of wide-range IPG strips. Thirteen distinguish induced samples and not in the control samples, whose isoelectric points （pI） were 4.0~8. 0.