Hello! I am wishing to a western blot for a protein that is expressed extracelluarly in an adherent cell line. Is it ok to use trypsin when I collect my cells? Or will it destroy the protein? Should I use a dissociation buffer? Thank you!
I would avoid trypsin because of the possibility of proteolytic cleavage of the protein. You could scrape the cells off the surface. You might also try washing the medium off the cells with saline, then detaching the cells with saline + EDTA.
Scrape the cells in existing medium while the dish is on ice. collect medium+cells, centrifuge and discard sup. Then proceed for lysis.
If I understand the question correctly, you are looking for a protein that is RELEASED by your cell line extracellularly? if that is the case, then all you need to do is collect the culture medium after certain period of culturing the cells and look for your protein in the media. Collecting the cells has nothing to do with your extracellular protein in that case.
However, if you are planning to measure the expression of a RECEPTOR on the cellular surface for that extracellular protein, then I would suggest not to use trypsin because it is a bit harsh on the cells and may strip off your receptor, better use EDTA in that case. You also need to bear in mind the sub-cellular localization of this "receptor" if you envisage to do a Western blot using whole cell lysates, as the "receptor protein" may be localized in other sub-cellular compartments, not only on the cell surface. So you may need to prepare subcellular fractionation before you do your Western blotting.
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