I am about to measure the secretion of a protein into the medium in a cell culture experiment (via exocytosis or extracellular vesicles). However, we have a certain level of death (10-20%) in our culture. Now the problem is how to discriminate between proteins that actively secreted and proteins that passively released due to death or death induced shedding. Normal cytotoxicity assays show the death rate, but do not give any clue about the impact of death on shedding and protein release. Is there any experience using a marker or an intracellular protein that we could employ as an indicator? My cells are neuron.
Dear Amir,
Though I am not aware of any assay which can discriminate that. But from protein list, you will come to know that whether the identified protein has a secretory signal sequence or not and then you can discriminate.
Second, there was a very nice paper in which the authors tagged the secretory proteins and they were able to harvest 100% pure secretory proteins byla belling. I saw the paper a month back and now couldn't find it on internet.
Though I will recommend you that the cell death (10-20%) is too much for any exosome based assay in your samples. I would recommend you to collect exosomes in really healthy conditions where cell death is 1-2% or even below that. That is acheivable.
Hope it helps!
Dear Ajay,
Thank you for your response. Unfortunately, the death in our cells are not due to trivial technical problem but innate properties of these cells and in some case related to pathologically susceptible phenotype. We try to reduce the death rate, however, still we are not completely successful. That's why I am looking for an idea to discriminate between two possible ways. The protein is not part of the conventional secretory pathway, but has been found in the extracellular medium, probably associated with synaptic activity. Recognition of protein is not problem since several different antibody is developed. The main aim is to find the mechanisms involve in release.
Dear Amir,
If the protein is known then I think its comparatively easy to track the release. You can tag the protein with GFP/RFP/YFP and then do the timpelapse microscopy which can tell you clearly if the protein is released and also may help to uncover more mechanistic insights.
If you are interested in more than one proteins than iTRAQ might be helpful but for single known protein, time lapse microscopy will be best.
Hope it helps!
Dear Amir,
I also want to add that you can also see if your protein is secreted actively from cells or enclosed in exosomes. Exosomes carry lots of proteins and RNAs. If it is enclosed in exosomes, then you can isolate exosomes by kit based method and do the protein isolation and characterize the same by western blotting. You should get the band if it is secreted by exosomes.
Hope it helps!
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