I measured the expression of a protein that is a receptor in a panel of cell lines. When I measured the protein levels by Western blot the protein levels were about the same in all cell lines , however when I measured the receptor levels by flow cytometry some cell lines had very low levels of the receptor. How it could happen?
Thanks Siamak. At the molecular/regulation level what is going on?
Hi,
I'm not a FACS specialist but what do you measure with the flow cytometry, is it the amount of receptor at the surface on intact cell or a more global protein abundance? For sure the WB will allow you to detect the whole bulk of the synthesized protein in the cell distributed throughout trafficking stage and only a portion of it may be in function at the plasma membrane. (I'd say this is not very optimal in terms of resource use but the cells in culture are in nutrient excess conditions and likely do not manage energy the same way cells in physiological conditions do)
Do you use the same antibody for both techniques, the antibody properties (affinity/avidity) can be very different for folded proteins in the FACS study vs denaturated polypeptide in the WB this happens in WB/IHC vs WB comparisons too.
Naive answers, hope this helps
Best
Daniel
Hi Pablo,
My suggestion to use Flow cytometry because it's a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations, and analyzing cell size and volume.
Regards.
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