Hirano H
Department of Molecular Biology, National Institute of Agrobiological Resources, Ibaraki, Japan.
J Protein Chem. 1989 Feb;8(1):115-30. doi: 10.1007/BF01025083.
Electroblotting method employing a semidry blotting apparatus for the subsequent protein microsequence analysis (Hirano, 1987) was improved. This method is convenient and allows rapid and efficient transfer of the proteins from a polyacrylamide gel (1 mm thick) onto the Polybrene-coated glass-fiber sheet or polyvinylidene difluoride membrane filter in only 20 min. The electroblotted proteins could be sequenced directly with the gas-phase protein sequencer at a 20-pmole level. This method was applied to the sequence analysis of winged bean seed proteins. A portion of the crude extracts from only one-twentieth of a seed of the winged bean was separated by two-dimensional polyacrylamide gel electrophoresis and electroblotted, and the N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of about 60% of the blotted major proteins, including nine Kunitz trypsin inhibitor-like proteins with heterogeneity in the N-terminal sequences, a protein that has a homologous sequence to the leghaemoglobin, nitrogen-fixing root nodule-specific protein, and a soybean basic 7S globulin-like protein could be easily identified.
采用半干印迹装置用于后续蛋白质微序列分析的电印迹方法（Hirano，1987）得到了改进。该方法简便，能在仅20分钟内将蛋白质从1毫米厚的聚丙烯酰胺凝胶快速高效地转移到涂有聚凝胺的玻璃纤维片或聚偏二氟乙烯膜滤器上。电印迹的蛋白质可直接用气相蛋白质测序仪在20皮摩尔水平进行测序。该方法应用于四棱豆种子蛋白的序列分析。仅用四棱豆一粒种子的二十分之一的粗提物的一部分，通过二维聚丙烯酰胺凝胶电泳进行分离并电印迹，然后分析印迹蛋白质的N端氨基酸序列。约60%的印迹主要蛋白质的序列能够轻松鉴定出来，其中包括9种N端序列具有异质性的Kunitz胰蛋白酶抑制剂样蛋白质、一种与豆血红蛋白具有同源序列的蛋白质（固氮根瘤特异性蛋白质）以及一种大豆碱性7S球蛋白样蛋白质。
