采用免疫胶体金标记结合负染色电镜技术对体外组装的中间纤维定性

Combining Immunogold Labeling and Negative Staining Techniques to Characterize Intermediate Filament Assembled in Vitro

将免疫胶体金标记与电镜负染色技术相结合，清晰显示体外组装的波形纤维具有１０ｎｍ中间纤维结构特征，抗波形纤维蛋白－金颗粒特异地标记在波形纤维上。用抗波形纤维蛋白－金颗粒对体外组装的微管进行标记则呈阴性反应。实验结果表明将免疫胶体金标记与电镜负染色技术相结合可以同时显示体外组装的细胞骨架纤维的精细结构和蛋白属性。

Using negative staining techniques, we showed that vimentin, expressed in bacteria, can assemble 10nm intermediate filament in vitro. Combining immunogolod labeling with negative staining techniques,we showed that the 10nm filament assembled in vitro can be labeled with anti vimentin antibody protein A gold particles specifically. Meanwhile, the microtubule assembled in vitro can not be labled with antivimentin antibodies. The above results demonstrated that both ultrastructure and protein components of cytoskeletal filament assembled in vitro can be characterized clearly at same time by combining immunogold labeling and negative staining techniques. Our results also suggest that it is a powerful technique in the electron microscopy study of macromolecules.