I'd like to measure enzymatic activity in E. coli and I need to perform a cell extract first. Can anyone share a nice protocol to do it?
Lysis buffer: ((50 mM Tris-HCl pH 7.5 - 100 mM NaCl
1 mM DTT (for intracellular proteins) - 5% glycerol (possibly)
1 mM EDTA - 1 mM PMSF - protease inhibitor))
Procedure: add 1ml to the bacterial pellet, resuspend and incubate in the cold room with rotation for 1 hour, after that sonicate or shear with G-Needls, centrifuge for 15 min @ 13000rpm, discard the pellet and keep the supernatant on ice.
You can always try to check for activity in situ with cell extract (even whole cells without disruption if your substrate can manage to pass the cell wall barrier). First thing you need to do is check for the known characteristic of yor enzyme target, i.e. does it have inhibitor or activator (you may consider to exclude or include them) at what condition is it active: what pH, ionic strength, even type of buffer. Having this info on hand, then you can resuspend your cells in the appropriate buffer containg all the ingredient needed for the reaction (apart from the substrate and other identifying chemicals of course). Just lyse the cell by sonication at mild condition to ensure no effect on your protein ( normally ten times ten seconds, with interval of 40 seconds, on ice). As negative control, you can provide the same cells that contain none of your enzyme interest, treat in the same way (paralel if possible). One thing that you have to keep in mind is, checking activityin a crude may lead you to a detection of small activity, because bunch of contaminants are still existing and your enzyme concentration may be small. I hope it helps.
Thanks so much, I'll take it in account and think about the best choice! :)
Additionally to MoHaMaD's buffer you can add DNase and lysozyme to remove the large amounts of DNA and cell wall from the bacteria. You can change the pH to your enzymes optimum, or to a buffer suitable for purification. Instead of sonication, which can damage some cell components, you can freeze the cells in liquid nitrogen and thaw them in room temperature water three times before centrifuging. This is particularly good if you have lots of different samples, as it can be done on lots of tubes at the same time. Also there is a product called BugBuster which rapidly lyses cells. It depends on your protein, as protease inhibitors and DTT may affect activity.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction