My question is focused on systematically troubleshooting liposome composition and detergent for cryoET analysis. I attached the paper by Yao et al 2020 (PNAS) where they began this process, but were interrupted due to COVID. I also attached the supplementary file to reference FigS1 panel E, where the liposomes composed of E. coli polar lipids were disrupted in the presence of DM detergent. I'm interested in troubleshooting detergents to use with EC polar lipid liposomes to get the uniformity and integrity they achieved with POPC lipids in DM detergent (Fig 1B/E).
I am not in a cryoET lab, but we have a close collaborator who is and will help with sample prep, image acquisition, and data analysis.
To me, it seems expensive and time-consuming to screen liposome composition/detergent combinations by cryoEM. Can I screen the liposomes by TEM instead? Or will the TEM process be too disruptive to tell if the liposome/detergent combination will be useful for cryoEM analysis?
Thanks in advance
Well, you will need to try, but I see no reason why it wouldn't work. The only inconvinience is the low concentration needed for negative stain, and wheather the population you 'll be observing is representative of the whole thing or not. But you will be having a better contrast (lower resolution) and it must be enough to check the integrity of liposomes, even though you can use other biochemical methods to caracterize them.
Jorgaq Pata Thanks so much! You bring up some good points. I think if I make a few grids per sample I might get a better idea of the population. I just wanted to be sure that negative stain TEM would show that the liposome population is homogenous in size and integrity but I wasn't positive that I would be able to see that by TEM because of the dehydration etc.
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