Can anyone tell me what would be a wise way to perform an experiment to check for the phosphorylation of protein in the cell lysates? I am treating my cells with melanoma exosomes and would like to see the phosphorylation status. Would it be wise to check this in serum free media or in normal physiological conditions (exosomes depleted FBS)?
There are phosphoprotein detection kits like:
https://www.lifetechnologies.com/order/catalog/product/P33300 and http://tymora-analytical.com/products/pimago/
I have used them succesfully, but Pro-Q expires quite soon.
Hi:
If you want to detect global change of phosphorylation, at least three methods you can apply: 1, anti-pan-phospho-antibosies by western blot 2, mass spectrometry for the phosphoproteome 3, Phospho-antibodies arrays. Many companies also provide convenient kits for detecting phosphorylation, please check it (http://www.sigmaaldrich.com/life-science/proteomics/post-translational-analysis/phosphorylation/protein-phosphorylation-analysis-tools.html). For your references please find in attachment, you will find some cases and more detail information.
Alternatively, if you just want to know whether the protein is phosphorylated, but not interested how many sites or which sites, you can use two more crude, but more economical methods. Both methods require your samples to be analysed with Western blot. One, treat your samples with calf intestinal phosphatase. The second method, run your samples on a PhosTag gel. Both methods are based on the principle that phosphorylated proteins migrate differently/slower on the polyacrylamide gel than non-phosphorylated proteins. Good luck!
Oh, and for Western blot analysis, it would be wise if you wash your samples with serum free media as serum contains proteins that may serve as unspecific binding sites for your antibody.
@Richard, But when I want to treat my cells with exosomes, do I treat them in serum free media or normal culture media. If I treat them in SFM, then I will loose more information regarding what happens in Physiological states in normal culture media.
Ofcorse while taking the lysates the cells are washed with PBS to remove the media and serum :)
HI,
I would treat the cells in normal medium and just wash them good while harvesting. Popele in my lab tend to take TBS instead of PBS for harvesting as well as for washing the blots. Don't forget to add phosphatase inhibitors. Do you want to have a general look on pjosphorylation or do you have a look on certain proteins? for a general look I woul also advice mass spec and for the latte one specific Abs
Regards
Sven
Hi,
As long as you wash your lysate you should be fine. I always did my treatments in culture media and I never encountered problem. Hope that helps. Let me know if my answer is not clear or you have more questions. Good luck!
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