I have been freezing my protein on ice using vitrobot and a blot force of -2. Even though the ice thickness looks good in atlas magnification. Most of my grid squares have no ice in the centre holes and only in the periphery. I use humidity 100% and 4°C. Does anyone have any suggestions about this?
My buffer contains very small percentage of detergent whi
ch is essential for my protein. I have attached atlas image and also a grid square image (the grid square is the one which has the pointer in the atlas magnification).
if I read the preview image right you are using UltrAu grids. Then, let me understand better, what is the issue? you get very thin ice in the middle of the hole, like a crown? Or you get completely empty holes in the middle of each square?
Anyway, that would be a sign of incomplete wetting of the grid.
What are the glow discharge settings?
What is the wait time used at the Vitrobot?
have you or the EM staff checked whether the blotting pads are properly aligned (that is, properly centered and not with the left one lagging behind)?
Yes they are indeed Quantifoil R 1.2/1.3 Gold grids.The squares are indeed completely empty in the centre.
We have an in-house glow discharge and we glow discharge for almost 20 secs and it has been tested with other samples like apoferritin and it works. I do not use any wait or drain time before freezing.
We have also checked the alignment of the blotting pads and they are also proper.
Cool, being UltrAu means they likely dont need any cleaning or pre-treatment. Then I would try adding an incubation time to allow proper spread of the sample drop (30,60, 90 well it depends on the viscocity, once with a compound in DMSO i had to go to 5min!).
Desperate attempt: i had a project where it kinda got impossibile to have sample inside the holes. So I applied 2ul on back and 2ul on front, waited 60 seconds and then blotted. It worked. Give it a try and let us know
