Hsieh J C, Lin F P, Tam M F
Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Anal Biochem. 1988 Apr;170(1):1-8. doi: 10.1016/0003-2697(88)90082-6.
A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.
已开发出一种用于分离蛋白质以进行微量测序的新方法。蛋白质通过在聚丙烯酰胺平板凝胶上进行等电聚焦分离。凝胶中的两性电解质用3.5%（v/v）高氯酸洗脱，然后将蛋白质电印迹到未修饰的玻璃纤维片上。用考马斯亮蓝染料染色检测玻璃纤维片上固定的蛋白质。然后从该片上切下蛋白质条带并插入气相测序仪进行直接测序。它们也可用十二烷基硫酸钠缓冲液提取以测定分子量。牛血清白蛋白、β-乳球蛋白A和大豆胰蛋白酶抑制剂已用作测试该技术的标准蛋白质。使用该技术，我们已确定了从鸡肝中分离出的一种酸性（pI 5.6）谷胱甘肽S-转移酶的部分N端序列（26个残基）。
