In vivo proline dehydrogenase microtiter plate assay
1. Grow 200 l overnight culture in microtiter plates in shaker/spec 37C (NCE succinate or appropriate medium).
2. Subculture 20 l into 200 l fresh NCE succinate (+/- proline or other inducer).
Medium contains ammonia as a nitrogen source, succinate as a carbon source and inducer.
Succinate is a poor carbon source so CRP is active which is necessary for full induction of the put operon.
3. Grow subculture to mid-log phase.
4. Transfer to a clear microtiter plate for OD600 reading.
5. Spin down plate at 4C, 2500 rpm for 15-20 min.
6. Resuspend pellet in 50 l cacodylate buffer.
7. Add 3 l toluene.
8. Shake for 10 min.
9. Add 10 l reaction mix containing O-aminobenzaldahyde.
10 Incubate with shaking for 45 min taking OD450 at 5 min.
11. Stop reaction with 2 l 10% TCA
12. Transfer to clear microtiter plate to read final OD450.
Grow overnight and subculture in clear microtiter plates.
Transfer to resistant microtiter plate to perform reaction (because of toluene).
Transfer stopped reaction back to clear plate for final reading.