目的 以课题组纯化获得的蛋白样本为观察对象，对比原子力显微镜(atomic force microscope，AFM)及扫描电子显微镜(scanning electron microscope，SEM)的观察结果，并总结AFM观察生物大分子的主要问题和解决方法。方法 将蛋白样本使用PBS稀释至15 nmol·L-1，分别固定于载玻片、硅片和云母片上烘干，制成固相观察样本，SEM样本在观察前镀铂，使用AFM及SEM观察蛋白质表面结构，计算样本高度，对比结果差异。结果 带正电的蛋白样本在观察时由于AFM探针的斥力会向右偏移;云母片能很好地消除蛋白正电荷从而避免样本移动;PBS能为蛋白样本提供良好的稳定环境，但PBS盐结晶会干扰探针运行和成像清晰度;SEM样本需要镀铂后观察，无法达到AFM的精度。结论 使用AFM和SEM均可在体外环境直接观察蛋白质结构，AFM能提供更高精度的观察结果;在蛋白样本稳定性允许的情况下首选超纯水为溶剂载体，乙醇等挥发性液体也可作为溶剂载体，AFM的应用可为药理学生物大分子互作研究提供一新途径。
Atomic force microscopic observation of surface structure of purified protein molecules in vitro
Aim To compare the observation results of atomic force microscopy(AFM)and scanning electron microscopy(SEM),and to summarize the main problems and solutions of AFM in observing biological macromolecules,using the observa-tion subjects of protein samples purified by our research group.Methods The protein samples were diluted to 15 nmol·L-1 with PBS,fixed on glass slides,silicon wafers,and mica sheets,dried,and made into solid-phase observation samples.SEM sam-ples were plated with platinum before observation.The surface structures of proteins were observed using AFM and SEM,sample heights were calculated,and differences in results were com-pared.Results Protein samples with positive charges tended to shift to the right during observation due to the repulsion of the AFM probe;mica sheets could effectively eliminate the positive charge of proteins to avoid sample movement;PBS provided a stable environment for protein samples,but the crystallization of PBS salts interfered with probe operation and imaging clarity;SEM samples needed to be plated with platinum before observa-tion and could not achieve the precision of AFM.Conclusions Both AFM and SEM can directly observe protein structures in vitro,with AFM providing higher precision results;when protein sample stability permits,ultrapure water is preferred as the sol-vent carrier,and volatile liquids such as ethanol can also serve as solvent carriers.The application of AFM offers a new approach for pharmacological studies on interactions between biological macromolecules.
atomic force microscopyscanning electron micro-scopereceptor structureprotein structures in vitroultrastruc-turemica carrier
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维普
