Bacterial Periplasmic protein localization
Posted 08 February 2005 - 04:48 PM
I have been desperately looking for a B-lactamase- alkaline phosphatase fusion systems or vectors. I have a bacterial protein that was predicted to be periplasmic and would like to prove it experimentally.
Have anyone done similar experiments that he or she can share?
Posted 09 February 2005 - 08:31 AM
Posted 21 February 2005 - 11:16 PM
expression as a periplasm localising fusion protein does not prove the point because you have artifially induced the localization with an appended signal.
National Chemical Laboratory
Posted 24 February 2005 - 03:37 PM
Posted 02 March 2005 - 09:11 AM
My protocol is similar with above.
(conc. of Tris-HCl and Na2EDTA can be adjusted.)
1.Resuspense 50mL cell pellet with 0.4mL fractionation buffer, waver at RT for 10min.
2.Centrifuge at low temperture,12000rpm,10min,throw supernatant away
3.Add 0.4mL MgSo4 5mM, votex 10 min to mix
4.Centrifuge at low temperture,12000rpm,10min,periplamic protein is in the supernatant.