This protocol is for labeling proteins expressed from an inducible expression vector.

1. Grow cells transformed with the expression vector in M9 minimal medium supplemented with antibiotics and 50 pg/ml of all amino acids except methionine. Grow overnight at 37 °C.

If using ³⁵S-EXPRESS, also omit cysteine.

2. Dilute 1:10 to 1:50 the overnight culture into fresh M9/amino acids minus methionine medium. Shake for 2-6 hours at 37 °C until cells have entered middle log phase.

3. Induce protein expression (e.g., by adding IPTG).

4. Incubate at 37 °C for 5 minutes.

5. Add 0.2-1 mCi ³⁵S-methionine.

6. Label for 1-10 minutes.

7. Add nonradiaoactive methionine (500 pg/ml).

8. Chill on ice.