Negative-stain spreading of protein-protein and protein-lipid complexes for EM
Brian Peter, McMahon Lab, Neurobiology Division, MRC Lab of Molecular Biology
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5% Uranyl Acetate in diH20
Carbon-coated collodion grids (purchase or see protocol)
Self clamping/locking forceps
100 ul PCR tubes
Clean glass slide, and Petri dish
Whatman paper, parafilm
Protein and liposomes of interest in same buffer
Negative staining is an extremely quick, easy method for visualizing protein, lipid, DNAÑnearly any macromolecular complex. It is useful for larger proteins, complexes or continuous oligomers (e.g., peptide or amyloid fibers, though the individual subunits are very small, can easily be visualized). The theoretical MAXIMUM resolution of negative stain is 7 ; this is nearly as good as cryo-EM, and much, much easier. More typical resolution is 15-30. It is generally possible to visualise complexes of several hundred kD (NB: simple calculations and experience indicate that it is NOT possible to see individual proteins, or complexes smaller than 200 kDÑin my hands, GroEL 14mers (840kD) or clathrin triskelia (600 kD) can be made out on a good grid but HSP60 monomers cannot). However, the major disadvantage of negative stain is that the protein is fixed in a low-pH, high ionic strength emulsion or layer of uranium ions. This does not necessarily cause major artifacts for all proteins (few artifacts are found for those proteins which have been looked at by both cryoEM and negative stain) , but basically, the effect on proteins is unknown. Liposomes or lipid tubes tend to collapse, probably from the high ionic strength during drying.
An alternative stain to UrAc is Phospho tungstic acid, or PTA. This can be made up to 2% in diH2O and then pHÕd to 7. The advantage of PTA is that the pH is more physiological and for some things the resolution may be greater; the disadvantage is that the contrast is less and I often had problems with big stain crystals, so it was hard to find a nicely stained area of the grid. But it makes a good control. If the structures look the same in both PTA and UrAc, they are unlikely to be a staining artifact.