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Cryo- and negative staining EM

We set up some grids of our protein for both negative staining and cryo-EM. There are a different types of grids, but a typical example can be seen here with a pen for scale. We were using copper grids coated with carbon for the negative staining, and gold grids with gold foil (UltrAuFoil) developed by Dr. Lori Passmore and Dr. Chris Russo here at the LMB for the cryo-EM. The grids have to be made hydrophilic, which is done by either glow-discharge, shown here, or plasma treatment (see my recent Twitter post for this). Either way you get a cool looking violet light. Very scientific. Samples are added to the grid and this is basically where the paths diverge for the two methods. For negative staining, the sample is stained with uranyl acetate. This allows you to see the shell of the protein, but not internal structures. All of this is performed at room temperature, as is the actually microscopy. For cryo-EM, there is no stain, and internal structures are visible. As the name implies, very
low temperatures must be maintained throughout the cryo- process. Samples are added to the grid and blotted so that they are in a very thin film of water. The samples is then plunged into liquid ethane (yes, liquid ethane, smoking is strongly discouraged here) at -181C. You can see the special container for the ethane here, with the small well in the middle holding the ethane, and the surrounding bath containing liquid nitrogen. At the top you can see the robot in action. Protein is spotted, blotted and plunged into liquid ethane. I have also stuck in a picture of me checking out our negative grids (which was not, unfortunately, as massive a success as I was hoping for).