In vivo proline dehydrogenase microtiter plate assay

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In vivo proline dehydrogenase microtiter plate assay

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.

Notes:
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.

Link: http://www.sci.sdsu.edu/~smaloy/Research/pdf%20files/Pro_DH_in_vivo_-_microtiter_.do.doc