Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition.
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 has achieved great success in studying structures of proteins and macromolecular complexes. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation. In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.
Graduate Group in Biophysics, University of California San Francisco, USA.
David S
DS
Booth
Agustin
A
Avila-Sakar
Yifan
Y
Cheng
D000818:Animals; D019843:Archaeal Proteins / Q000032:analysis / Q000737:chemistry; D005293:Ferritins / Q000032:analysis / Q000737:chemistry; D006736:Horses; D046911:Macromolecular Substances / Q000032:analysis* / Q000737:chemistry; D008854:Microscopy, Electron / Q000379:methods*; D016624:Negative Staining / Q000379:methods*; D009707:Nucleosomes / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry
D019843:Archaeal Proteins; D046911:Macromolecular Substances; D009707:Nucleosomes; D011506:Proteins; D005293:Ferritins; D046988:Proteasome Endopeptidase Complex