So I transfected a mammalian cancer cell line with a protein of interest, which is usually a surface protein.
Now I wondered whether I can detect the shedded protein via flow cytometry, and if so, how (Ab coupled to beads?)
Also I thought about doing an intracellular staining to check whether the protein is expressed, but not shedded. Would that work with cell permeabilization (saponins) and a transport inhibitor (brefeldin/monensin)? Or how would I approach this question?
I know I can also do it with WB, but I think FACS might be faster.
Thanks already for an answer :)
Flow cytometry would have the advantage that you can find subpopulations of cells that act funny. Otherwise, if you do not have a method to stain for your protein of interest on cells, use WB for a trial. That is so much more robust and sensitive.
Dear Stefan,
You can definitely do FACS for your sample. If you want to check the surface expression by FACS then just don't permeabilize your cells. Incubate the antibody (coupled to fluorescent marker or bead labelled) with cells as such. However also check the surface expression of a protein that you know is expressed on the surface of that cell line (if you have the FACS ab for it), this is just to ensure the technique. In case you want to look at the presence of expressed protein then permeabilize your cells (according to the preferred method of permeabilization for your cell line). Use a transport inhibitor.
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i agree with all researchers and article here will help you
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870581/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499300/
Hey there,
first of all thanks for all the answers,
I might have been not completely clear with my question, so the protein of interest is an artifial soluble form of a membrane protein, we just added a signal peptide, so that it will be shedded into to cell culture media (so that we can purify it for Crystallography)
And I was wondering, whether I could detect the proteins in the SN via FC, I guess this bead array would maybe make sense then.
To check whether the signal peptide is dysfunctional (since we couldn't detect our protein in the SN with WB) I thought about the ICS, for future experiments, since I think FC is faster and more elegant than WB.
I've only done ICS to stain cytokines, so I wasn't sure whether I could stain literally any intracellular protein.
Dear Stefan,
You can stain intracellular protein as well as nuclear proteins and get them acquired on FACS. In case of clinical samples when you have lesser amount of starting material researchers prefer doing FACS for intracellular protein than western blot. It is faster and requires lesser number of cells.Fixing, permeabilization and staining procedure is modified accordingly for cytoplasmic signaling proteins as compared to cytokines during ICC. If your protein is in SN then perform bead array or ELISA.
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