35S labeling of proteins

1. Wash cells (e.g., 35 mm plate) twice with methionine-free tissue culture medium.

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

2. (optional) Starve cells in methioine-free medium for 1-16 hours.

3. Incubate cells in methionine-free medium + 35S-methionine (~50 mCi per sample).

4. Incubate for 1-18 hours.

5. (optional) Chase with normal medium for 1-2 hours.

6. Remove medium.

7. Wash twice with PBS.

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

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

10. Analyze using SDS-PAGE or 2D-gel.