I am working on identifying phosphorylation site (s) within a novel protein and and their roles. It appears through mass spectrometer of my protein that there are phosphorylated serines, threonines as well as tyrosines. I am planning to validate all these sites using western blot with an anti-phospho antibody for each amino acid. I am curious to know if anyone has experience with detecting phosphoserines vs phosphotyrosine etc and can comment on which ones are easier to detect easily.
I personally prefer phospho-tyrosine. However, make sure you maintain the conditions your protein get phosphrylated by similar to you mass spec tested in order to have consistent and reproducible results. Otherwise detecting phosphorylation sites might be challenging.
I do regularly western blot for Serine, Threonine, and Tyrosine phosphorylated groups and so far the main difference is in the antibody itself. I would check literature first and see which antibodies (if any) have been already used in western blot, they usually are the most reliable ones.
Ass Bassam mentioned above pan-phospho-tyrosine antibody is very good compared to pan phospho-serine or threonine antibodies. On the other hand, If you have specific phospho-sites to be analyzed, you can get good motif specific antibodies against pS/pT. For example the substrate motif antibodies from CST worked good for us in case of many proteins (Even though there are some site preferences and batch variations)
The pan- phosphotyrosine antibodies like 4G10, pY-10 and pY-100 are more stable and reliable than the antibodies for detecting phospho-serine or phospho threonine. The important thing is to ensure that you retain the phosphorylation during cell lysis and during the process of western blotting till the time you throw in the primary antibodies. Sometimes, I have used a mixture of the 4G10, pY-10 and pY-100 antibodies and have obtained better results, since each of these antibodies detect slightly different motifs with varying specificities. Since all of these antibodies are monoclonal, secondary detection is not an issue. More important is the relative ratio of these antibodies and their dilutions that you will have to work out empirically.
Best of Luck!
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