A few additional details are important for helpful suggestions. Are you planning an in vitro experiment ? Do you plan forced expression of your factor in tissue culture? Are good antibodies available?

Right now I am planning for Invitro . I want to treat the TF with a growth factor. Good antibodies are available. Thank you.

If your plan is to treat cells with a growth factor you may isolate proteins with and without treatment and perform a Western of a 2D-Gel. If this works there are probably a few candidate sites. You may mutate them and add a tag, transfect them and analyze again.

Very first, try to find out what is already deposited in databases!

A surprising amount of data has been entered in publicly available databases, (NCBI.org) often containing information that has never been formally published (from mass spec experiments). Look for the latest protein GenBank (.gb) entry of your sequence, it will contain the known information in the annotation section there you may get a first impression on whats known, which can save a lot of unnecessary work . Then you can go ahead and mutagenize the site and investigate your biology. Also Mass Spec is a fast way to map a site, it may also reveal a lot of additional information on other PTMs of your protein of interest:

• DOI: 10.1016/S0076-6879(02)51853-X

Good luck! Thomas :-)

Thank you a lot Thomas Martin Sternsdorf and Uwe Borgmeyer for your answers. I will be asking more questions if you allow.

I would also recommend to check existing databases for already known phosphorylation sites, and then take it from there. For example:

https://www.phosphosite.org/homeAction.action

Besides this, the suitable strategy might be directed by available equipment and methods established in your lab. Mass spec of samples from non-stimulated vs. stimulated cells can help you to identify related phoshosites if you have access to such lab and can isolate sufficient amount of your protein.

For phosphorylation assay in vitro, you would need purified target protein (or a fragment containing your phosphorylation site) and purified active kinase.

Thanks, Eugen Werwein . If I say " How do you determine if a particular protein has phosphosite and determine where it’s located on the protein? what will be the best answer

Not sure, there is THE best answer to this question, really depends on the overall context.

I would say, if a protein has serine, threonine or tyrosine amino acids, then there are chances that these residues might get phosphorylated.

Yes that has serine,therione or tyrosine amino acids. Would you like to suggest me some journals to read about this. Eugen Werwein

Thomas Martin Sternsdorf Hi Thomas, I have to do the assay using western blot. I have the antibody of the transcription factor. Would you like to help me to design the assay? Please also suggest articles like the one you shared about mass spectroscopy.

Happy to help, it has been a while, if you still need help, mail me at [email protected]

Dear Md Abu Siddique,

to convincingly detect phosphorylation by western you will need to optimize your run as much as you can, to reproducibly see the small shift it induces in mobility, which is almost always a hard sell to reviewers. I am happy to share protocols, but let's do this by email and not here, too tedious and I will be easier reachable, your message is from February and I only saw it now.

So in the future better at: tsternsdorf(at)gmail.com

that should expedite things. Send me a mail if this is still relevant and I gladly do my best to help.

Thomas :-)