Negative stain protocol (Rini lab)

'''If frozen as 20 uL aliquots at -80C, the Uranyl stain can last forever. If you see crystals on the grid, you didn't wick the stain well enough. It is not the stain. '''

Copper-Rhodium grids

 * M300-CR
 * M400-CR (most frequently used grid size)

Gridbox

 * 71155

Tweezers

 * Dumont style 5, fine tip(0.06x0.1 mm will be OK), non-coated. Example: 0303-5-PS-1 (electron microscopy sciences @ Amazon.com)
 * Quantity: 2


 * Reverse action, anti-capillary tweezers 0.10x0.15 mm. Example: 5378-NM Do not get the gold plated ones or the super-thin ones!
 * Quantity: 2

Chemicals

 * Acetone (For cleaning the grids if desired. Can also be used to dissolve the nitrocellulose membrane to recover grids from failed attempts of making continuous-carbon grids.)


 * Uranyl formate (With our protocol, only tiny quantities will be used for each grid. One bottle shared among labs should last years.)
 * Uranyl is a toxic heavy metal in a easily digestible salt form. So use proper caution and try minimising waste.


 * 2% Uranyl formate can be made with 0.2 g Uranyl formate and 10mL H2O in a 15mL tube. The water only needs to be clean. No heating, No NaOH, just shake in a tightly closed 15mL tube for the stain to dissolve and a quick and gentle spin to bring down the drops from the cap. After filtering through a .22 um or .45 um syringe-filter (hydrophilic, the same ones for proteins), the 2% stain can be saved forever at -80C as 20 uL or larger aliquots.


 * Upon use, adding 1V water to the stain to make 1% stain can further reduce both the consumption of stain and chance of getting crystals on the grid. Adhere to a concentration (0.75%, 1%, 2%) based on your personal preference though.


 * Uranyl formate is not as light-sensitive as legends tell. Working under room lighting or staying on open bench for 1 hour will both be fine. The frozen aliquots will be stable at -80C for at least 1.5 year with no observable change. If black crystals are observed on the negative stain specimen, it is most likely due to insufficient wicking (therefore very high local concentration at end of drying), not the stain being "old".

Glassware
A round, glass lunch box, with a diameter of 8-15 cm and a smooth rim.

Making amorphous carbon grid
1. Fill a round glass container with clean water. Ideally slightly overfill the container so that the water top surface is convex in shape.

2. Use a 200uL pipetman, drop a 5-10 ul drop of collodion on the top of the water. Wait for 5 min till the amyl acetate solvent evaporates and a solid film can be detected by gently poking and dragging. This film is formed from the nitrocellulose dissolved in collodion.

3. Lay M400-CR grids with the silver(shinnier) side facing the nitrocellulose film. If you let the "wrong" (less smooth) side facing down, there is going to be ... no problem. When one has 20-100 grids on the film it is enough. Beginners should only lay 20 grids or so each time.

4. Take a piece of the backing paper of parafilm, or similar "waxy" paper. First lightly wet one side of the paper by touching a water surface, then tap this side on a dry filter paper to remove the excess water.

5. Lay the semi-wet paper over the grid-on-nitrocellulose film floating on water. Wait for the paper to attach to the NC film. If deemed necessary, cut the extra film outside the paper with pipetman tip so that the film won't fold back onto the grids. Then lift the paper carefully from one side. The film and grids should adhere to the paper and be lifted.

6. Lay the paper with grids on bench (grids facing up ofc). Put some 1x3 inch glass slides (or anything with some weight) on its edges to help holding it immobile. Let it dry (a lamp over it can help, but do not put the bulb too close - the vicinity of a incandescent bulb is at a few hundred °C). Now we have an assembly of NC film-grids-on-paper, from top to bottom.

7. Go to a carbon evaporator, evaporate as much carbon as you like to the top of the NC film. The grids are now ready to use. Very thick carbon is not a problem for negative stain. Too thin carbon may have problems adsorbing the protein (or surviving glow discharge).


 * If unsure, the presence of the carbon film can be checked under light microscope, especially when some squares have broken films. The carbon film is actually quite strong. If a grid is sonicated under water, many squares will have broken carbon films but they will still cling to the grid.
 * One may also put some water on the grid surface and observe under light microscope to see the water drying - at the end of drying, in each square one can see water retracting from the centre along the film surface, instead of suddenly burst.

Negative stain Procedure
Before start: clean the work area, put one or two pieces of large (10-20 cm diameter) filter paper on the intended work area to catch, if any, spillage. Tear a few stripes of filter papers for wicking the grids later. Fold each of these small pieces once so that they can stand on the large filter paper.

1. Glow discharge the grids for 15-60s according to your preference. As long as the solution spreads on the grid with no help it is enough. It is advised to use the glow discharged grids within an hour. However, using them after 3 hours didn't seem to be a big problem.

2. Grip one grid with an anti-capillary tweezer. Lay the tweezer on top of a large piece of filter paper. The carbon side of the grid faces up.

3. Put 2-5 uL of protein (5-15 ug/mL concentration, needs to be tested for new proteins) on the grid, wait for 10-60s. No need to be very exact. Most binding reactions follow a roughly parabola-shaped curve. Most binding occurs in the first few seconds, everything later accounts for the rest 10%.

4. Blot away the protein solution on the edge of one of the filter paper stripes prepared earlier.

5. Wash the grid with 3-5 uL of liquid. This liquid could be any of the following (they all work!):


 * Water
 * Saline(150 mM NaCl)
 * TBS
 * The uranyl stain

6. Blot away the wash solution. No need to be too thorough here.

7. Put 2-3 uL of 2% Uranyl stain on the grid. Leave for 15-60s. Blot off the stain as much as possible (with a good filter paper) but do not scratch the carbon surface (lightly touching the surface with some furry edge of the paper is fine). Tip: The thick Whatman filter paper works best; Whatman papers have their wicking rates stated on the box.

8. Leave the grid to dry either on the tweezer or in the grid box. Do not worry about the light.