My protein has a pI of 4.1 and I am purifying it in Hepes(10mM)NaCl (150mM) buffer. When I freeze my protein sample for cryoEM using vitrobot, I dont see any particle. I glow-discharge my grid first before applying protein sample. I believe, I see no particles because of the highly negative charge of my protein. How should I get rid of this issue Would adding non-ionic detergent help ?
Dear Hirdesh
considering the composition of your buffer I assume you are talking about a soluble protein. Detergents could help, but your ice would become less electron transparent. A thin backing of amorphous carbon could also do the trick, but again, your contrast would become much worse.
In our recent pre print we show how the presence of hydrophilized graphene monolayers deposited on regular grids allows particle adsorption to the graphene-water interface.
This dramatically improves the stability of our protein (the fungal Fatty Acid Synthase), and is also ideal contrast-wise because graphene is a single continuous monoatomic layer, with signal is invisible up to 2Å frequency.
The preparation of these grids is very easy and reasonably cheap.
You may want to give a look:
"The deadly touch: protein denaturation at the air-water interface and how to prevent it"
full text available:
https://www.biorxiv.org/content/early/2018/08/25/400432
Preprint The deadly touch: protein denaturation at the water-air inte...
Best,
Davide
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