I am carrying out experiments with cell secretomes ( collected after incubating with one type of cell for a few days). The secretome is again used to study the effect on cancer phenotype. Before proceeding with expensive methods such as lipidomics and proteomics, I was wondering is there a way I can separate either lipids/ proteins from the secretome but can also be used for further cell treatment.
Thank you
Hello,
Sure but need some more info!
So essentially you are using spent-media from old cultures to supplement the whole thing into a new cultures to see some bioactivity, right?
What's your interest, proteins or metabolites/ lipids? Got to between different approaches based on the interests.
For proteins its easy, harvest the media, precipitate everything with acetone/ ethanol and wash multiple times and redissolve the pellet to do proteomics on them, or supplement the protein pellet into the cell-type to see that in deed it's the proteins that are showing activity.
For metabolites slightly tricky but doable. Lyophilize 1-5 mL of that "active media" and extract polar metabolites in 80% methanol or say extraction solvents using protocol: https://www.protocols.io/view/metabolite-extraction-and-derivatization-of-plasma-723hqgn/guidelines Redissolving this extracted metabolites in some PBS or so, can help recheck the bioactivity on cell-type as well, to confirm that it's the metabolites that are bioactive!
For lipids, prior to lipidomics, just take the media (after filtering out the cells of course!) and extract in excess amounts of MTBE (Matyash protocol) :Article Lipid Extraction by Methyl-Tert-Butyl Ether for High-Through...
and perform lipidomics.The extracted lipids can be redissolved in say
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