I belive 1000 PSI will work but yield will be very low. I have used the method of Rosen, B. P. (1986) Methods Enzymol. 125, 328-336. Shortly:
Grow 0.5 L E. coli to OD600~0.8
Collect by centrifugation 10min, 10000g 4C.
Resuspend in TSC buffer and centrifuge as above.
Weigh the pellet and resuspend in 5 volumes/weight of TSC buffer.
Add MgCl2 to 2.5mM final
Add DNase to 1 ug/ml final (important!)
Add DTT to 0.5mM
French Press 4000 PSI twice.
Remove whole cells by centrifugation as above.
Ultracentrifuge soup from above 260,000 g, 1h, 4C
Suspend pelet in TSC
TSC buffer:
140mM Cholin Chloride, 10mM Tris-HCl pH=8.0, 250mM Sucrose.
Yoram
BTW, 20,000 PSI gave mixed orientation membranes so preassure is important...
Thank you very much Dr.Gerchman.Previously I tried 1000psi and activity was coming very less which laid to thought that lower psi might be causing problem in the assay.I really appreciate it.I will try and update you with the result.
- Badges
- Science topic
- Similar topics
- Engineering
- Chemical Engineering
- Membranes