牛脑中神经特异性S-100蛋白的纯化及鉴定

牛脑充分匀浆后经三次硫酸铵分级沉淀,再通过一次DEAE-Sepharose CL-6B层析柱,线性梯度洗脱后共收集4个峰洗脱液。PAGE分析(7.5％凝胶)显示第3峰为单一区带;免疫双扩散证实该洗脱液中蛋白为S-100蛋白。SDS-PAG E分析显示S-100蛋白分子量约为10kD;非还原条件下,凝胶过滤(Sephadex G-75)显示S-100蛋白位于MW为20kD区域。认为该纯化方法简便、快速,可获得较高纯度的S-100蛋白,活性高达1∶128以上,完全能满足进一步研究之用。

Isolation,Purification and Characterization of A Nervous System Specific S-100 Protein from Bovine Brain

Fresh bovine brains were homogenized immediately. The crude proteins were extracted by repeated ammonium sulfate precipitations at 50, 80 and 100% saturation, and they were absorbed on a DEAE-Sepharose CL-6B column (1.3×13cm) by FPLC. The S-100 protein was eluted by a linear gradient (0—0.6mol/L NaCl). PAGE (7.5% gel) of S-100 protein showed a single band. Immunodiffusion result has identified the immunoactivity of the purified S-100 protein (3rd eluted peak). SDS-PAGE by Phast System showed the S-100 protein with a MW of approximately 10kD, and Sephadex G-75 filtration indicated S-100 protein eorresponding to a MW of 20kD under non-reducing condition. We conclude that this method is efficient to obtain enough pure S-100 protein with immunoactivity for further studies.