I did my SILAC experiment (Lys8/Arg10) with bone marrow derived macrophages (cells were cultured for 4 days in light or heavy medium). My cell numbers were quite low (100 000 cells), but I could get a decent amount of proteins according to coomassie staining. After analysing with Maxquant and Perseus, I only got a small number of proteins and all of them were Bovine. I guess these are coming from the FBS?
Could anybody help me troubleshooting?
How are you extracting your protein? And what are your washing procedures to remove FBS?
I wash with PBS and extract as described inGeiger T, et al., Nature protocols (2011)
It could depend on how closely you're aspirating to the pellet once you wash in PBS and how much lysis buffer you're adding. If you're only adding a small amount of lysis buffer and there is residual PBS (plus serum) left at the bottom then this could be a factor contributing to your problem. You of course do not want to aspirate so close to the pellet that you're losing cells but it is a bit of a balancing act. Maybe using more cells and therefore more lysis buffer accordingly may help you as well as id there is residual FBS this will be further diluted to a much lower amount. Maybe try doing 2 washes in PBS?
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