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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If questions, contact Brian: bpeter@mrc-lmb.cam.ac.uk or Harvey: hmm@mrc-lmb.cam.ac.uk

 

Materials needed

5% Uranyl Acetate in diH20

Carbon-coated collodion grids (purchase or see protocol)

Self clamping/locking forceps

100 ul PCR tubes

Glow-discharger

Clean glass slide, and Petri dish

Whatman paper, parafilm

Protein and liposomes of interest in same buffer

 

1. Prepare protein, buffer, tubes and grids. Place grids onto a cleaned microscope slide. Stick down clean Parafilm on lab bench; place wetted Whatman paper on either side (to minimize drying).

 

2. Mix protein and lipid in PCR tubes. Total volume will depend on how much protein you have to use. Usually I mix at least 20 ul; otherwise evaporation can drastically change the salt content.

 

3. Glow discharging of grids: Place slide with grids on top (carbon side up) in vacuum chamber, and pull vacuum and glow discharge. If not using automatic timer, glow discharge for 30-60 s. After chamber is vented, place slide into petri dish and return to lab. If there is no glow discharger available, this step can be omitted; however, the results will not be as clean (less protein will bind, and maybe more grot on the grids).

 

4. Pipette drops of UrAc (20-40 ul each) onto ParafilmÑtwo drops per sample. Have one piece of (folded) Whatman paper to hand to absorb excess stain and wash buffer from grids during staining protocol. Have another piece of Whatman, labeled, onto which stained grids will be placed.

 

5. Hold a grid in clamping forceps. Pipette 6 ul of protein/lipid solution onto grid. After 20-45 s, blot off buffer-protein solution onto Whatman paper, touch to first UrAc drop, blot off excess stain immediately, touch to second UrAc drop. Wait 10-30 s, then blot off UrAc (take care to blot out liquid trapped in forceps). Grids can then be washed by touching to a drop of buffer and blotting off the excess. Place grid onto labeled Whatman paper

 

6. Allow grids to dry for a few minutes, then place in grid box (note positions/IDs in lab book). Grids can be visualized immediately, and are usually stable for months or years.

 

 

 

Background/comments

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.

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