Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition.

摘要: Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology. In recent years, this method achieved great success studying structures proteins and macromolecular complexes. Compared with cryomicroscopy (cryoEM), which protein samples are embedded thin layer vitreous ice, staining is simpler sample preparation dried heavy metal salt to increase specimen contrast. The enhanced contrast stain EM allows examination relatively small samples. addition determining three-dimensional (3D) structure purified complexes, can be used for much broader purposes. For example, easily visualize obtaining information such as homogeneity/heterogeneity the sample, formation complexes large assemblies, simply evaluate quality preparation. video article, we present complete protocol using an observe negatively from preparing carbon coated grids acquiring images microscope operated at 120kV accelerating voltage. These protocols have been our laboratory routinely followed by novice users.