I am trying to extract proteins from small cut sections of frozen human cerebellum. I was used standard protocol involving RIPA buffer and sonication and protein yield was good. However I need to get rid of lipids and since I am working with small volume (Less than 100 ul), I cannot follow the standard protocol for whole tissue.
Any advice on how to get rid of lipids without losing proteins?
Thank you.
The RIPA buffer contains both IGEPAL and sodium deoxycholate, both detergents which will solubilize lipids. If you insist on going lipid-free, I'd suggest lysis in a simple PBS-buffer without those detergents, followed by ultracentrifugation to pellet the lipid vesicles. Though you'll lose the membrane proteins this way, as they either cling to the lipids or denature and aggregate into insolubles. Beyond these, you'll inevitably lose some proteins to isoelectric aggregation, as no detergents are present.
You could also try solvent-washing with i.e. phenol to extract the lipids, if you don't require your proteins to be still "alive" for your purpose. There are protocols for total protein extractions, which are rather nuclear approaches.
https://www.nature.com/articles/nprot.2014.022
If you just need them in solution, you can resuspend the aggregated protein in 4M guanidinium chloride or >=8M urea. In case the proteins stay suspended (no clear solution) then you can detangle them by putting them at pH13 for a few minutes, followed by neutralization.
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