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.