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Can anybody tell me more about detection of phosphoserine/threonine in stimulated cell lysates by Western blotting? I want to use phosphoserine/threonine antibody but I do not know whether I should use monoclonal or polyclonal or whether I have at all any chances to get results by this method and should better use 32P-labeling of cells instead?
Thanks!
A monoclonal antibody tends to be more specific to an epitope. It is usually time consuming and somewhat expensive to generate them. Then, you need to test several to find the best one.
If one is available commercially, then you can order it - but depending on your application you may encounter problems in sensitivity. Technical support staff can help, if you are pursuing a commercial antibody.
Polyclonals can give as strong or stronger a signal, but obviously the background will be higher.
If you need to resort to radiolabelling then it strikes me that you aren't seeing a high degree of phosphorylation to begin with.
I do not mean to generate antibody by myself. My question was actually about whether there is any sence to buy general anti-phosphoSerine or anti-phophoThreonine antibody to test for phosphorylation on these amino acids in the protein I work with. I did not find examples in the PubMed that people go this way, so I ask here. Will such strategy work or it is better to use radioactive labelling (I try to avoid using radioactivity, but if there is no choise I'll do it)?
I've only used anti-phosphotyrosine (commercial). It worked well.
Radiolabel --- not done this in the context you suggest. Wouldn't all the phosphate - containing groups become labelled. You wouldn't know if your signal is from phosphoserine, phosphotyrosine, phosphothreonine, or other. I suggest finding a vendor and asking tech support. Their job is to know which applications work well.
Sorry I could not be of more help.....