I’m performing some cellular pharmacokinetics and need to separate cytosol from vescicle content in a pellet of cells treated with our compound, to determine its on-cell walk and compartimentation.

I’ve found out that I can obtain a good cytosol lysate by simply adding 40-50uL (per every 2*10^6 cells) hypotonic buffer with mgcl2, Hepes buffer and saccarose, and incubating pellets for 30mins on ice.

How can i treat the remaining “membranaceous” pellet to recover the compound that entered endosomes by uptake or got stuck inbetween the double layers? I’ve read that freeze-thawing cycles break vescicles as well. Is it true?

Is soft sonication a good technique?

I know many protocols use SDS or similar detergent but I’ll run my samples on LC-MS so I should avoid surfactants.

Thank you so much!

  • Similar topics
  • Heat
More Lorenzo Tagliazucchi's questions See All
Similar questions and discussions