Hey,
I am not an Silac expert, but as far as I understand the method you pusle your cells with heavy amoni acids for a short time. Therefore a tryptic digest for splitting afterwards woul affect all already labeled proteins of your extracellular matrix and also cell surface proteins. For example it is advised not to take trypsin if you want to FACS analyse adherent cells for surface markers. Taken this into account I would rather not trypsinize the cells. But by the way why do you want to do this? I guess you just culture the cells shortly after SILAC pulse right? so why to passage them?
Regards
Sven
In theory the trypsin-EDTA can contain amino acids that could interfere with your SILAC labeling. I still use it though and don't think that it is a great problem, simply use as little trypsin as possible to be on the safe side. For each cell line that I've used I've determined the efficiency of incorporation of the SILAC amino acids (you should always test this prior to doing the experiment itself) and I get incorporation of >96%.
Hi Sven,
Thanks for your answer. You are correct in saying that it might affect cell surface proteins.
BUT, in SILAC, Not for a short pulse, rather for at least 6 generations cells are to be grown in order to have all the proteins labelled with the heavy amino acids. Therefore, We need to passage cells in between like in usual cell culture, before they are ready for downstream analysis, usually Proteomics-MS Analysis.
Thanks, Emmely. Yeah, theoretically, it is usually suggested not to use. But, practically, experienced people say that it doesn't affect much. Although, one is always hesitant Initially in doing so.
Thanks,
Divyank
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