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Microscopy Laboratory Techniques

High resolution negative staining

(From Valentine et al, 1968. Biochemistry 7:2143-52)

Rationale: For the highest resolution with negative staining, there should be little or no support film, but some support is necessary to hold the protein. In this method the proteins are supported by a carbon film and "embedded" in a film of uranyl acetate. The film is cast on mica, which provides the cleanest possible surface for the carbon.

  1. Freshly cleave a piece of mica, coat with a carbon film using the vacuum evaporator.

  2. Put ~30 mg/ml protein solution in a small vessel.
  3. Cut the mica to 3-4 mm2 pieces. Hold a piece with forceps and push into the solution of protein at a 30-45 degree angle. Do not let the film detach completely from the mica.

  4. Let the film sit on the protein solution for 20-40 seconds.
  5. Pull the mica back and allow the film to sit on the mica.

  6. Then slide the mica and film onto a solution of 1-2% uranyl acetate or uranyl sulfate. Pick up the film with a copper grid, dry, and examine in the electron microscope.

Variations: A more stable film can be obtained if you pick up the film on a grid coated with a holey formvar film.

A wrinkled carbon film may be better than a smooth one, as it seems to keep pools of uranyl acetate around the protein better than smooth films.

Try varying the protein concentration for optimal staining. The proteins should be closely spaced to collect the stain but if they are too crowded proteins will be overlapped and will not be easily resolved.

The carbon support film should be as thin as possible (3-10 nm) and should be prepared on freshly cleaved mica. Once the carbon coated mica has been prepared it can be stored in a dessicator almost indefinitely. Thicker support films will lower image contrast.

Stain solution should be removed from below the surface of a stock stain solution to avoid contaminants from precipitates that may exist at the surface or the bottom of the stock solution. The precipitates do not affect the overall quality of the staining procedure but will result in local heating of the support film when exposed to the electron beam, which in its most benign form will result in drift, and at its worst will result in a ripping and curling of the support film.

The carbon support film should only be exposed to proteins that exist in the bulk solution and must not be exposed to denatured material that is usually present in the meniscus.

The greatest potential source of contamination and the one most often ignored is the contamination that arises from the tweezers and the support grid. At no time should the tweezers or the support grid be dipped into either the protein solution or the negative staining solution.

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