Li X, Hatanaka K, Guo L, Tsushima M, Kitamura Y, Yamamoto A
Department of Etiology/Pathophysiology, National Cardiovascular Center, Osaka, Japan.
Thromb Haemost. 1993 Apr 1;69(4):331-4.
In plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added to normal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.
在血浆中，蛋白S以游离形式存在，并与C4b结合蛋白形成复合物。将¹²⁵I-蛋白S加入正常人血浆并进行SDS - 8%聚丙烯酰胺凝胶电泳后，凝胶的放射自显影片在游离蛋白S位置仅显示一条单带。基于此证据，我们开发了一种过氧化物酶染色免疫印迹法来定量人血浆中的总蛋白S，该方法包括十二烷基硫酸钠聚丙烯酰胺凝胶电泳，然后印迹到硝酸纤维素膜上，以及一种灵敏的抗生物素蛋白-生物素化过氧化物酶染色法（ABC技术）。通过免疫印迹法测定蛋白S具有可重复性，变异系数为7%。最低可检测到1 ng的蛋白S。C4b结合蛋白不影响蛋白S的测定。与其他免疫测定法相比，这种过氧化物酶染色免疫印迹法具有直接估计目标蛋白表观分子量、消除非特异性染色以及在不使用放射性同位素的情况下具有高灵敏度的优点。
