I realise this differs between cell lines so an exact answer is not possibly.
But if anybody could tell me the cells they work with and the exosome protein quantity they achieve it would help me optimise my protocol.
I'm currently ulracentrifuging 8mL of 48 hour cell culture media (serum free) from PC3M cells, which is eventually resuspended in 100uL RIPA buffer. Protein quantification suggested it was below the limit of quantification of the Lowry assay I used giving a result ~200ug/mL
Is this good bad or ugly?
Thanks,
Sam
I use MDA-231s and generally use much larger volumes. I incubate 75% confluent cells (in a T175) for 24 hours in SF DMEM prior to ultracentrifugation of the supernatant. I resuspend in small volume of NP-40 based lysis buffer and use a bradford to quantify - I am still optimizing my procedure but I generally get low yields - only 3-7ug in total from a starting volume of 100mL - typically around 0.1 or 0.2 ug/uL - so similar to what you got if you quantified at 200ug/mL - but you achieved this from 8mL! It seems like the extra day of incubation may help yield or your cell type produces more exosomes than mine. If you are hoping to get more protein, I would start with more conditioned media.
Thanks Will, I doubt I actually got that much, I think it was just an artefact of being so close to the LOD. So does NP-40 not interfere with a Bradford assay? I know some detergents do, which is what I assumed gave me a hint off a result with the Lowry assay as I use RIPA. Anyway, thank you, I will try increasing my volume and flask size/number.
Hi, just for your information Samuel, my yield is very close to Will's... I am working with ECFCs and I use regular RIPA buffer. To give you an idea I can get 0.5ug/ul starting from 60 ml of media, so any suggestion to improve this yield will be welcome!!
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