Preparation of Negative Stain Solution and EM Grids

Prepare negative stain solution

*** Uranium formate (0.75%) [light sensitive, aggregate if exposed to light; weakly radioactive?]
*** Uranium acetate

(1) Weight out 37.5 mg into a small beaker.
(2) Add 5 ml boiling distilled water, stir 5 min in the dark.
(3) Add 5M NaOH (~10 – 20 ul) until the solution becomes slightly dark yellow (too much NaOH precipitates the stain),
and stir for another 5 min in the dark.
(4) Filter the solution with a 0.2 um syringe filter into a falcon tube wrapped with aluminum foil. Mark the tube with date.

Stain proteins on EM grids

(1) Apply (25-30 ul / drop) 2 drops of distilled water and 2 drops of stain solution on parafilm.
(2) Apply 2.5 ul protein sample to a glow-discharged EM grid coated with a continuous carbon film.
(3) Wait 20-30 seconds to allow protein adsorption.
(4) Blot the grid from the side with a piece of filter paper (dont' let it dry).
(5) Quickly touch and blot the grid for 1st water → 2nd water → 1st stain drop.
(6) Keep the grid touching 2nd stain drop for 20-30 seconds, blot, and wait until it is dry.
(7) Store the grid in a grid holder.