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Star Republic: Guide for Biologists

35S labeling of protein in bacteria

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.