Dear all, I have an issue figuring out good plunging conditions for large lipid structures: I have carbon coated C-Flat 2/2 400mesh copper grids with large (elongated) lipid structures on it. The length is several µm up to mm, spanning not only many holes, but many squares. Prior to the freezing with a Vitrobot, the grids are (and have to stay) fully emerged in solution. This means, that when I take the grid, a small film of solution is already present at both sides of the grid. I have tried many different ways for freezing the cryo gids but the ice is still too thick to see anything. If I'm lucky, there is one square that I can see, but here the ice has vanished almost completely. I tried blotting for 1,2 and 5 seconds, blotting manually from the side of the grid and both combined, but the ice remains too thick. Any input is highly appreciated. What can I still try to reduce the remaining water? I guess blotting even longer or multiple time might work, but then I'm afraid my structures are getting blotted away or destroyed. Did anyone use more extreme setting? Blotting twice or longer? Thank you all for the help. Best Dario
Hi Brack,
In my opinion, you may try an ultrastable holey gold alloy film (45 nm) It’s a derivative of the patented C-Flat holey carbon films. Also, dropped the small amount of protein sample to adjust the condition of humidity. It might help you to improve grid preparation.
Hello Dario,
You could try several things but as you mentioned if you are not sure of the Sizes of your structures, you could do a study using a 200 or 300kV scope as you did not say the HT of the scope you are currently using. Also if your structures are as large as you say, why not try Phase Contrast Light Microscopy, I commonly did this to look at multi Phase Liquids and Gels before I had access to Cryo-TEM or as a first step in a study.
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