3.2 Uranyl Formate
(see Note 4)
As with Uranyl acetate, it is important to minimize light exposure
of UF during the following steps. Final UF solution is best used
fresh and made fresh when required
1. Weigh 37.5 mg Uranyl Formate.
2. Place in 10 mL beaker (containing a stir bar) and cover with a
larger beaker wrapped with aluminum foil.
3. Add 5 mL of just boiled Milli-Q or double-distilled water.
4. While covered, stir gently for 5 min.
5. Stop stirring and add 6 μL of filtered 5 M NaOH.
6. Wait for ~15 s and continue stirring in the dark for an additional 5 min.
7. Filter the solution using a 0.22 μm filter and 5 mL syringe.
8. Solution should be yellow in color without precipitation.
9. Store at room temperature in the dark for 3–7 days or until
precipitation starts (see Note 5).
3.3 Staining Protocol
Protocol below is based on starting with a high concentration of
protein.
The protocol can be adjusted for this and other types of samples (see Note 6).
1. Prepare all your starting material, including any sample dilutions, preparation of stain, and organization of the negative
stain area before any of the next steps.
2. Place grids carbon-side up on a glass slide (wrapped in parafilm
for easier handling) or aluminum grid block. Glow-discharge
the grids at default vacuum for ~30 s at 15 mA (see Note 6).
Stain grids within ~1 h of glow-discharging.
3. Place 3 drops of 50 μL Milli-Q water on the surface of a large
sheet of parafilm for every grid to be stained. Keep the areas per
grid separate and leave space between the drops for slight
movement. For samples containing detergent, add 3–10
drops of Milli-Q water and adjust use accordingly (see Note 7).
4. Place 2 drops of 50 μL UF or UA on the parafilm for the
current grid to be stained only. Perform steps 5–10 below
immediately following step 4 to minimize the exposure of
stain to light.
5. Using anti-capillary tweezers, pick up one grid from its edge
being careful not to bend the grid. Place 3 μL of sample on the
carbon side of the grid (see Note 8) and without the tip of the
pipette touching the grid.
6. Wait for 5 s and blot the excess sample by touching the side of
the grid to a piece of filter paper briefly (see Note 6).
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Shane Gonen
(see Note 4)
As with Uranyl acetate, it is important to minimize light exposure
of UF during the following steps. Final UF solution is best used
fresh and made fresh when required
1. Weigh 37.5 mg Uranyl Formate.
2. Place in 10 mL beaker (containing a stir bar) and cover with a
larger beaker wrapped with aluminum foil.
3. Add 5 mL of just boiled Milli-Q or double-distilled water.
4. While covered, stir gently for 5 min.
5. Stop stirring and add 6 μL of filtered 5 M NaOH.
6. Wait for ~15 s and continue stirring in the dark for an additional 5 min.
7. Filter the solution using a 0.22 μm filter and 5 mL syringe.
8. Solution should be yellow in color without precipitation.
9. Store at room temperature in the dark for 3–7 days or until
precipitation starts (see Note 5).
3.3 Staining Protocol
Protocol below is based on starting with a high concentration of
protein.
The protocol can be adjusted for this and other types of samples (see Note 6).
1. Prepare all your starting material, including any sample dilutions, preparation of stain, and organization of the negative
stain area before any of the next steps.
2. Place grids carbon-side up on a glass slide (wrapped in parafilm
for easier handling) or aluminum grid block. Glow-discharge
the grids at default vacuum for ~30 s at 15 mA (see Note 6).
Stain grids within ~1 h of glow-discharging.
3. Place 3 drops of 50 μL Milli-Q water on the surface of a large
sheet of parafilm for every grid to be stained. Keep the areas per
grid separate and leave space between the drops for slight
movement. For samples containing detergent, add 3–10
drops of Milli-Q water and adjust use accordingly (see Note 7).
4. Place 2 drops of 50 μL UF or UA on the parafilm for the
current grid to be stained only. Perform steps 5–10 below
immediately following step 4 to minimize the exposure of
stain to light.
5. Using anti-capillary tweezers, pick up one grid from its edge
being careful not to bend the grid. Place 3 μL of sample on the
carbon side of the grid (see Note 8) and without the tip of the
pipette touching the grid.
6. Wait for 5 s and blot the excess sample by touching the side of
the grid to a piece of filter paper briefly (see Note 6).
120
Shane Gonen