A eukaryotic protein is predicted to be secreted by the SignalP 4.0. And how can I determine wether the protein is indeed secreted or not by the experiment? Is there any simple assay can be done to test its secreted? Thank you!
Collect the conditioned culture medium from the cell culture. Centrifuge out any cells. Test the supernatant for the protein by Western blot or ELISA or mass spec. If the cell culture is grown with fetal bovine serum, it may be necessary to remove the albumin first.
The options suggested by Adam are correct. I might like to add that many membrane proteins (receptors etc) also contain a signal peptide since they rely on the secretory pathway to reach the cell surface. In such a case you might want to include FACS analysis to your list of experiments.
Thank you! Adam and Ruben. My protein does not have any tag, so maybe I can't detect its secretion by Western Blot, unless I prepare the monoclonal antibody of my protein. So is any assay like laboratory-based E. coli alkaline phosphatase (PhoA) assay to test the secretion of eukaryotic protein. Or do you think the E.coli alkaline phosphatase(PhoA) is also suitable for eukaryotic protein secretion? Thank you!
Dear Qiang Zhang,
the easiest way to test your hypothesis might be having a cDNA coding for your protein synthesized with a C-terminal tag. Then express it in suitable cells and check if you can find it in the supernatant and fractionate your cells to check if it gets somewhere else (and it's also nice to see the unprocessed protein still containing the signal).
If you really want to raise an antibody, don't do monoclonals, it's much to tedious and expensive and not worth the effort. There are tools to predict sites with good antigenic potential, then have a peptide synthesized and have a polyclonal made against it.
If the protein is an enzyme, then you can test the medium for the activity of the enzyme.
I agree with Wolfgang that making an anti-peptide polyclonal would be faster, easier and cheaper than a monoclonal.
Another experimental approach is pulse-labeling and immunoprecipitation. You radiolabel the cells for a short time with [35S]methionine, then test for the presence of the protein in the medium at various time post-labeleing by immunoprecipitation, SDS-PAGE, and autoradiography.
In any case, it would be valuable to have a second protein as a negative control. This would be a cytoplasmic protein. This is to control for the possibility that your protein is a cytoplasmic protein that got released into the medium when cells died.
Be sure that your cell viability is very high because you don't want to see the protein in the media due to cells breaking open and releasing cytoplasmic materials. Material in the media should have the signal peptide processed off as an indication of secretion. You could also inhibit the secretory pathway and look for accumulation in the cells.
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