I want to know whether a new protein is phosphated by other kinases or not. And there is no such phosphorylation antibody to detect, so how could I know whether the protein could be phosphorylated and which site(s) is(are) phosphorylated? Is there any simple method by which I can prove it ?
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You can use a prediction site to have an idea (http://www.cbs.dtu.dk/services/NetPhos/) and here you have a description of other methods (http://www.rndsystems.com/mini_review_detail_objectname_MR08_ProteinPhosphorylation.aspx). Hope this help.
I'd recommend putting the protein sequence into Scansite (http://scansite.mit.edu/). It's a great way of identifying potential phosphorylation motifs for a huge variety of kinases.
Practically speaking, it depends how much money you have. You could confirm that it's phosphorylated at all by blotting the protein using a site non-specific phosphorylation antibody such as 4G10 (detects phosphotyrosine). If you want to identify specific sites though, with no prior knowledge, I'd recommend going down a mass spectrometry route if you have the money.
I used the GPS tool for prediction. It's helpful try them with your query protein and choose the kinase options based on your requirements.
There are many phosphorylation site database ,which can solve your problem ,for example ,GPS, Phospho ELM, Phospho plus ,etc
The prediction sites mentioned by others are nice. Phosphosite takes it a step further and tells you any phpsphorylation site ever actually observed in mass spec studies, so there is already some evidence the site exists:
http://www.phosphosite.org/
Firstly, thanks to Madhurima. Secondly, when I use the phospho-Tag antibody to detect the phosphorylated site, how to make sure that I can distinguish a very weak phosphorylated protein? Should I do the IP? Moreover, how to differentiate the same size proteins at the similar( or the same) position if I use the total preteins in WB?
In addition to the excellent suggestions already made you should consider that Ser/Thr phosphorylation may not be detected so easily as Tyr phosphorylation by western blot because there are to my knowledge no really reliable pan-pS/T antibodies around. In case you have the required facilities available, you could also consider to do a radioactive in-vivo labelling experiment, IP your protein and check for its phosphorylation by autoradiography. Doing a phosphopeptide map subsequently would even give you information on which peptide is actually phosphorylated. There is also the possibility to use a panel of recombinant kinases to test whether your protein is a substrate for any of them - the company I am working for offers such a service called KinaseFinder - if you are interested you may get information on our homepage (see attached link)
http://www.proqinase.com/content/view/90
Our lab worked on this question for more than ten years. Please try http://gps.biocuckoo.org. The webserver could predict substrates and sites for over 400 kinases. Please contact me if you have any further questions.
gps.biocuckoo.org
GPS (Group-based prediction system) version 5.0 (2020). The webserver could predict substrates and sites for over 470 kinases.
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