Negative stain TEM

Single partilce

Negative staining is a simple sample preparation method in which protein samples are adsorbed to a continuous carbon film and embedded in a thin layer of dried heavy metal salt to increase specimen contrast. The enhanced contrast of negative stain EM allows viewing of relatively small biological samples (>100kDa).

This method is used mainly to determined 3D structure of small protein and protein complex (<200kDa), assess sample quality (e.g. purity and homogeneity) and determined initioal 3D structure using Random Conical Tilt (RCT) method. It can also be used to answer specific questions concerning protein-protein interactions and identify protein/domain assembly. antibodies or Fab can be visualized when bound to their antigen.

Drawbacks and Benefits of Negative Staining

Benefits

  • High contrast.
  • The sample is easy to prepare.
  • Almost no radiation damage.

Drawbacks

  • The resolution limit is between 15Å-20Å (the size of the stain salt grain).
  • Artefacts can arise if the stain is uneven.
  • Flattening of the sample upon drying of the grid 

stain				     pH range
-------------------------------------------------	
Sodium (K) phosphotungstate (PTA)      5-8
Uranyl acetate 			       4.2-4.5
Uranyl formate			       4.5
Sodium silicotungstate 		       5-8
Ammonium molybdate		       5-7
Methylamine tungstate		       6-7
3mm EM Copper grid
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Protocols

Negative Staining

Reading

Negative Staining and Image Classification - Powerful Tools in Modern Electron Microscopy.

EM Analysis of Protein Structure