Team:Freiburg/Protocols/Periplasmatic Preparation

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periplasmic preparation

periplasmic preparation by osmotic shock
remarks concerning the protocol (Pavel, Toolbox)

material: Tris, EDTA, sucrose, MgCl2 …
duration: 1,5h

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1. discard the supernatan and weigh your cells
2. resuspend cells thoroughly in ice-cold sucrose solution: 20mM Tris-HCl pH 8.0, 25% (w/v) sucrose and 0.5 EDTA. Take 8ml of sucrose mix per gram cell. Transfer the samples to 50ml Falcon tubes and incubate on ice for 15min. Note: This step creates a concentration gradient across membranes
3. spin down the samples at 10 000xg for 20min. Remove the supernatant (the sucrose fraction) and save it on ice.
4. Extract the periplasmic content by osmotic shock. Dissolve the pellet in 8ml per gram cell of a mix of ice-cold 5mM MgCl2 and half a tablet of protease inhibitor cocktail (Roche) or 0.5mM PMSF) and incubate on ice for 30min.
5. Spin down the samples at 10 000xg for 20min. Remove the supernatant and mix it with sucrose fraction. Supplement with NaCl and Imidazole to desired concentration for purification.

protocols, protpur