I've been looking at various protease inhibitor cocktails and it's a bit frustrating that when I'm lysing CHO cells I need such a small volume of protease inhibitor cocktail that I would need a thousand aliquots of it to avoid repeat freeze thaw cycles. I've been thinking, don't some protein extraction protocols involve solvents that inherently inhibit proteases by denaturing all proteins and thus proteases? For example, does Trizol not inhibit all proteases? Does TCA precipitation not render all proteins inactive, including proteases? Secondly, do protease inhibitors tolerate freeze thaw cycles of dissolved in pure DMSO? Or what about protease inhibitors at room temperature in pure DMSO?
Hi Adam,
There are different protease inhibitors that can be stored at different conditions (RT, +4C, -20C, etc). I am attaching one Guide for Protease Inhibition where producers explain how their inhibitors work and how they can be stored.
Thanks, I've looked at that before. Unfortunately that doesn't answer my questions.
Hi Adam, I don't know about trizol, but TCA precipitation is routinely used to stop proteinase activity in many assays. But I'm not sure that it permanently kills them.
Same goes for many solvents. They precipitate proteinases, but they may still show activity if the pellet is reconstituted.
Adding hot reducing SDS buffer will kill all proteinases. That will be messy, but the resulting snot can be cleared using small bore needle aspirations.
Unstable inhibitors (e.g. PMSF, Cl-methylketones, etc) can be stored in pure anhydrous DMSO at - 80oC. Freeze-thaw cycles should be OK if there is no water in the preparation.
Thank you very much Steingrimur! That helps tremendously. Do you think anhydrous DMSO can allow freeze thaw of all protease inhibitors?
Hi Adam. I'd like to bring you the following thesis abstract on the role played by solvents on enzymes inactivation. The work was carried out using a non destructive microwaves spectroscopy apparatus (in my former research laboratory).
Fennouh Souad Ph.D. thesis presented on June 2, 1997 at UTC (University of Technology Compiegne) France.
Optimization through organic solvents of acetylcholinesterase inactivation by paraoxon. Interactions study by micro-waves spectroscopy.
Abstract:
In aqueous medium the detection of paraoxon, an organophosphorus
pesticide, by acetylcholinesterase is not enough sensitive and does not fulfill the requirements of the European Union (1 ppb).
Acetylcholinesterase inactivation by paraoxon depends on the organic solvent used and on its concentration. This effect is more important on free acetylcholinesterase than on immobilized one.
Four organic solvents which maintain the catalytic activity of free acetylcholinesterase and enhance its inactivation by paraoxon were selected: dimethyl ketone at 0.05% (v/v), 1-propanol at 2.5% (v/v), 1-decanol at 1% (v/v) and cyclohexane at 5% (v/v). This last one gave the best efficiency on paraoxon detection (250 times compared to a reaction medium without organic solvent) which resulted in a paraoxon detection threshold 5 times lower than the value fixed by the European Union.
The analysis of the interactions that take place in the reaction medium by micro-waves spectroscopy consolidated the results obtained for free acetylcholinesterase. This technic also highlighted the importance of the interactions which happen between the aqueous phase and the organic solvents.
The organic solvents influence the conformation of the reaction medium in such a manner that the hydration of free acetylcholinesterase is maintained through the interaction with the polar parts of the reaction medium. The enzyme configuration is then optimal for the maximal inactivation by paraoxon.
If they are soluble in DMSO, I don't see why not.
But every time you expose the anhydrous DMSO to air there will be some ambient humidity that gets in. So to be on the safe side its probably best to limit the freeze-thaw cycles to around 10.
- Badges
- Science topic