Phosphorylation of protein - Trouble with backphospho (Sep/03/2004 )

Hi everyone!!

I was wondering if anyone of you ever performed backphosphorylation of a protein, and if so what kind of phosphatase and/or protease inhibitors you used.
The thing is that I am able to phosphorylate my immunoprecipitated protein without a problem. The next step for me was to try backphosphorylation wich consist of stimulating phosphorylation in vivo prior to immunoprecipitation and phosphorylation in vitro. Less radiolabeled phosphate should then be incorporated in my protein. But it is not the case. I tried phosphatase inhibitor cocktails, NaF, Na3VO4...

I'd really be grateful if anyone of you had any idea of why my backphosphorylation doesn't work, or if you think of something I could try...

Thank you very much

Simon

-Simonsays-

QUOTE (Simonsays @ Sep 3 2004, 02:31 PM)

The problem you may be encountering is that while your factor of interest is a substrate in vitro, it may not be one in vivo. I would recommend in vivo metabolic labeling with orthophosphate to confirm that it is a bona fide substrate.

-Ewen-

Hi, I have not a solution for your worry. Is only a question for you. You say that have not problem with the phosphorylationn assay. Could you tell me what is the method you use.

Thank you

Carolius

-Carolius-

Thanks Ewen for your answer... it is actually what I was thinking too... I'm doing the metabolic labeling next week as I just ordered the radiolabeled phosphate.

Carolius : 1- I immunoprecipitate my protein
2- I wash it and incubate it with phosphoserin, diolein, ATP, 10µCi 32P-ATP and 75-80ng of purified PKC for 5 to 15 minutes at 30°C. If you need more details, here's my email....

simonbousquet@hotmail.com

Simon

-Simonsays-