猪肺胞膜窖和窖蛋白分离纯化鉴定及其纳米结构的原子力显微镜研究

摘要

Caveolae are various invaginations on the surface of cell plasma membrane that related to signal transduction and disease.Caveolae which has unique characteristics of the flotage density ar separated from pig lung by treating with non-ionic detergent TritonX—100 which can not dissolve with caveolae.Gel electrophoresis shows the caveolae relative proteins.Determining the caveolin—1 by dot hybridization shows the caveolae contain caveolin.The protein content is 0.791 13 mg/mL by coomassie blue staining.This method to obtain caveolae and caveolin has some repeatability and stability,which can apply to any biological tissue and cell line.atomic force microscopy（AFM） shows caveolae is 79nm.Then,a new method for isolating caveolae rapidly and completely and identification caveolin by dot hybridization is established.%利用4℃条件下胞膜窖不溶于非离子去垢剂ThritonX—100以及所在膜区域具有独特的浮力密度特性,从猪肺中分离得到胞膜窖.用聚丙烯酰胺凝胶电泳检测到特征蛋白条带,利用特异性标志蛋白Caveolin—1进行点杂交实验鉴定信号蛋白,结果表明：胞膜窖中含有Caveolin蛋白.考马斯亮蓝法得到样品蛋白含量为0.791 13 mg/mL.原子力显微镜显示胞膜窖的大小为79 nm.该方法可应用于各种生物组织及细胞系,且得到的胞膜窖和Caveolin蛋白重显性好、结果稳定,由此建立了一种高效快速分离完整胞膜窖并用点