Kristensen D B, Inamatsu M, Yoshizato K
Yoshizato Morphomatrix Project, ERATO, JST, Kagamiyama, Higashihiroshima, Japan.
Electrophoresis. 1997 Oct;18(11):2078-84. doi: 10.1002/elps.1150181134.
The present study devised a new procedure for concentrating proteins cut from primary two-dimensional polyacrylamide gels prior to their structural characterization. Gel spots containing a protein were cut from several identical two-dimensional gels and loaded on the top of a high tensile strength stacking gel in a Pasteur pipette. The protein was concentrated electrophoretically into a small volume in the narrow tip of the pipette. The concentrated protein was then blotted from the stacking gel onto a polyvinylidene difluoride membrane, where the protein was subjected to tryptic on-membrane digestion. Utilizing this system, we identified 22 proteins chosen randomly from two-dimensional gels of proteins from rat dermal papilla cells by internal microsequencing. As much as 1.5 mL volume (cut gel spots in protein sample buffer) could be loaded onto the Pasteur pipettes, generally yielding a final on-membrane area of approximately 2 mm2 after elution concentration and electroblotting onto polyvinylidene difluoride membranes. We concluded that this newly devised system is effective and useful for concentrating proteins prior to structural characterization, and that larger volumes than previously recommended can be effectively concentrated, with little or no effect on the final on-membrane area occupied by the concentrated proteins.
本研究设计了一种新方法，用于在对从原始二维聚丙烯酰胺凝胶上切下的蛋白质进行结构表征之前对其进行浓缩。从几块相同的二维凝胶上切下含有蛋白质的凝胶斑点，并将其加载到巴斯德吸管中的高拉伸强度堆积凝胶顶部。通过电泳将蛋白质浓缩到吸管狭窄尖端的小体积中。然后将浓缩的蛋白质从堆积凝胶印迹到聚偏二氟乙烯膜上，在该膜上对蛋白质进行胰蛋白酶膜上消化。利用该系统，我们通过内部微测序从大鼠真皮乳头细胞蛋白质的二维凝胶中随机鉴定了22种蛋白质。多达1.5 mL体积（蛋白质样品缓冲液中的切下凝胶斑点）可以加载到巴斯德吸管上，洗脱浓缩并电印迹到聚偏二氟乙烯膜上后，通常最终膜上面积约为2 mm2。我们得出结论，这种新设计的系统对于在结构表征之前浓缩蛋白质是有效且有用的，并且可以有效浓缩比以前推荐的更大体积的蛋白质，对浓缩蛋白质占据的最终膜上面积几乎没有影响。
