Hello
which methods are good to disrupt the E.coli periplasmic space to extract protein and what is the mechanism of each method?
Thanks in advance.
- Osmotic lysis could be a good option, where cell pellet is treated with 0.5 to 1.0 ml chilled 5.0 mM MgSO4 solution followed by its incubation for 10 min at 4 oC. Centrifuge the pellet at 10, 000 rpm and use the supernatant as periplasmic fraction. The pellet can further be used to obtain the ctyoplasmic fraction after sonication.
Thank you.
But what is the ratio between the cell pellets amount and buffer volume and how to find it out? and how should i find out which concentration of buffer will have best effect on the method? because i did'nt find any relation between them in the papers i've read.
Do you know any published data for the method you have mentioned?
Best wishes.
Hi Nii, You may find the following paper useful :) Good luck!
http://www.ncbi.nlm.nih.gov/pubmed/20932056
The mention method relies on osmotic shock. Where cell are being permeabilized using chilled MgSO4. You may optimize the concentration of MgSO4 from 3 mM to 10 mM by taking pellet from 10 mL culture. In one go you may optimize it. Well I ll send the link of publication once I found.
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology