TCR Zeta Chain & Phospho-species - immunoprecipitation and WB (Dec/04/2003 )

Ennnnnnnigmatic !

I'm doing an immunopricipitation of the CD3zeta chain of the TCR and I see a good signal of the non-phosphorylate form of the chain (16kDa) in WB with the same Ab that I use for the IP.

However, I can't see the other phosphospecies of the zeta chain
(p21 and p23) with the CD3zeta Ab. But I can see them with an
Ab against phospho-Tyrosine. (It is also like that in many papers).

If I can immunoprecipitate all the phospho-species of the zeta
chain...(16, 21 23 kDa) How come I can not all see them with
the Ab. Why I can only see the 16 kDa.

If the epitope recognize by the Ab would change with the
phosphorylation...I shouldn't be able to immunoprecipitate it
........?!?!?!

I hope that I has been clear...
...PLEASE HELP me to resolve that paradigm!..

Thanks for time to answer me...

-Bill
Montreal, Canada.

Acturally, I have no idea of what you're working on and I have no experience on IP. But I have a possible explanation (perhaps not the case): the three different phospho-species are naturally together at least in some conditons,perhaps via noncovalent bonds. So you can IP all with the antibody against any of them. You can test with another antibody against one of them other than what you're using. You may also change the conditions to destroy the interaction. Or you can first do IP and then do some filtration to just allow smaller complex to go through and then probe on the WB.