32P labeling of phosphorylated proteins

1. Wash cells (e.g., 75-cm2 flask) twice with phosphate-free tissue culture medium.

If serum is required for cell growth, use water-dialyzed serum.

2. Incubate cells in phosphate-free medium + 32P orthophosphoric acid (2.5 mCi/107 cells).

Less radioactive orthophosphoric acid (0.1-0.5 mCi per sample) may be used for immunoprecipitation experiments.

3. (optional) Induce phosphorylation of target proteins.

4. Incubate for desired time (5 minutes to overnight).

5. Remove medium.

6. Wash twice with PBS.

7. Solubilize cells with solubilizing agent (e.g., buffers containing 2% SDS or 4% NP-40).

8. Centrifuge at the maximum speed to pellet cell debris.

9. Proceed to immunoprecipitation, SDS-PAGE or 2D-gel.