Jungblut P, Eckerskorn C, Lottspeich F, Klose J
Institut für Humangenetik, Freie Universität Berlin, Federal Republic of Germany.
Electrophoresis. 1990 Jul;11(7):581-8. doi: 10.1002/elps.1150110709.
Purification and chemical characterization of proteins may be achieved by combining two-dimensional electrophoresis (2-DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2-DE pattern as possible. Under optimized conditions, blotting efficiencies between 60% and 100% were obtained for five marker proteins; the mean values from four regions of a 2-DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60% and 79%. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patterns, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.
蛋白质的纯化和化学特性分析可通过二维电泳（2-DE）与微量测序或氨基酸分析相结合来实现。为实现这种结合，必须将蛋白质尽可能完全地从凝胶转移至测序仪中。在本研究中，使用疏水膜作为转移的支持物，通过半干印迹法将蛋白质从凝胶转移至膜上。对印迹条件进行了优化，以尽可能多地获得复杂二维电泳图谱中多种蛋白质的高印迹效率。在优化条件下，五种标记蛋白的印迹效率在60%至100%之间；来自小鼠脑复杂蛋白质混合物中29种未知蛋白质的二维电泳图谱四个区域的平均值在60%至79%之间。所比较的四种市售疏水膜在对全蛋白质模式进行印迹后，膜上的蛋白质量仅显示出细微差异，而单一蛋白质在其中一种或另一种膜上的含量更高。印迹优化的结果使我们能够提出一种印迹机制，利用该机制可以针对有问题的蛋白质系统地改进印迹条件。
