phospho protein - (Jul/29/2007 )

The protein with which I am working has many phosphorylation sites. In order to confirm this I was thinking to do phosph labelling of cells expressing protein of interest and immunoprecipitate it.
Can some one tell me the protocol for phospho labelling?
Or any better way to look for the phospho protein?

thanks

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well western blot may be better, don't you think so ?
But maybe not antibodies?
in that case produce an epitope tagged protein, and the purify it and mass spectroetry would be an alternative.

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phospho labeling of cells? what do you mean? offering P32 or P33 ortho-phosphate in medium??

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what I mean is
1. Transfect cells
2. grow them for 6 hr
3. starve the cells in phosphate free medium for 2 hr
4. add radioactivity which can provide hot phosphate (Yes P32 orP33 ortho phosphate I am not sure yet which one would be better)
5. collect the cells
6. isolate total protein and immunoprecipitate with antibodies against protein which I choose to express

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well seems correct. Just add NaF to all buffers or any phosphatase inhibitor you prefer.

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I do not understand why radioactive labelling is necessary; it is a big mess, if P32 or the more expensive P33, does not matter;

show phosphorylations by anti-phospho-Tyr, or -Ser or Thr antibodies; use phosphatase inhibitors as fred_33 adviced;

try to think of alternatives with shRNA/siRNA down regulation, toxins or receptor stimulation

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If I read you cerrectly you mean to add phosphotase inhibitor after collecting cells during immuno precipitation.
As some one else said I know it will be a big mess with P32/P33. But I think it still should be doable. What you think?

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it is good alternative. What about IP it and send for mass spec? It would be difficult to see enough protein.

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totally ! the inhibitor should e added directly from the time you start to disrupt cells, till the end. NaF is the common used or Naorthovanadate. There exist tablets too.
you need to contact mass spec facility to see how much protein you need. Then, well what i can say is to do a try and see if it's sufficent. You need to ask your boss/colleagues for the expected yield

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you can give a radioactivley labeled protein to Ms/Ms only after certain period of time till radioactivity is faded; in the case of P32 it is ca. half a year!

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