All cells contain a variety of intracellular proteases. This becomes a problem when one wants to make cell extracts for protein isolation since cell-lysis protocols invariably release any number of active intracellular proteases that can quickly hydrolyze proteins in the extract. Therefore, an efficient means of inhibiting a wide spectrum of proteases is mandatory for a variety of assays (e.g. western blotting, immunoprecipitation, reporter gene activation, gel shift, enzymatic activity assays etc). For this purpose, I have been using Complete Protease Inhibitor Cocktail Tablets from Roche for some years now. Since I started using this cocktail, I would never consider going back to the old-fashioned alternative of dealing with aliquots of different protease inhibitors (e.g PMSF, Leupeptin, Aprotinin, etc.).
The Complete Protease Inhibitor Cocktail Tablets are not a mere mixture of inhibitors - it is qualitatively different from the above-mentioned chemicals. The cocktail consists of a mixture of several protease inhibitors, both reversible and irreversible, with broad inhibitory activity specificity. It is worth noting that Pepstatin must be added to lysis buffer if aspartic proteases must be inhibited. For all other purposes the cocktail is ready to use in a very practical form – a tablet that is very water-soluble. It can simply be dropped in the lysis buffer a few moments prior to use.
The ease of use for this protease inhibitor cocktail is perhaps it’s greatest feature. It is just a matter of dissolving one tablet in the appropriate volume of buffer just prior to use – there is no need to do calculations or to weigh chemicals. In my experience, I have found this cocktail to be reliable because the level of protease inhibition is highly reproducible, constant and maximal in a variety of conditions. Also, it is safe to use since there is no need to handle toxic compounds or organic solvents and it is easy to store because the product can be stocked in a single vial at +4 C for a long period of time.
The product is sold in two concentrations (to make 10 ml or 50 ml of buffer) and is available in an EDTA-free form (to keep metalloproteases active when required). In cases where less than 10 ml of buffer are needed for a single experiment, the tablet can be dissolved in water to make a concentrated solution that can be stored at –20 C, thus avoiding waste. Although I have not personally used this cocktail in anything but mammalian cells, Roche claims that it is effective for plant, yeast and bacteria as well.
In conclusion, I would have not problem recommending the Roche Complete Protease Inhibitor Cocktail tablets to anyone. It is the most practical, easy and effective way to assure the complete inhibition of proteases when working with cell extracts.
Dario Coletti, PhD
Post-Doctoral Fellow
Mount Sinai School of Medicine