What are the most commonly used protease inhibitors?
The most commonly used protease inhibitors and their working concentrations are included in the table below:
|Protease inhibitor||Inhibits||Suggested working concentration||Stock solution (200x)|
|PMSF*||Serine proteases and cysteine proteases such as papain||85 µg/ml (0.5 mM)||17 mg/ml (200 mM) in ethanol or isopropanol|
|Leupeptin||Serine and thiol proteases||0.5 µg/ml (1 µM)||0.1 mg/ml (200 µM) in water|
|Pepstatin||Aspartic proteases||0.7 µg/ml (1 µM)||0.14 mg/ml (200 µM) in ethanol|
|Aprotinin||Serine proteases||1 µg/ml (0.15 µM)||0.2 mg/ml (30 µM) in water|
|Pefabloc®||Serine proteases||0.5 mg/ml (2 mM)||100 mg/ml (400 mM) in water|
|Na2-EDTA||Metalloproteases||0.35 mg/ml (0.75 mM)||70 mg/ml (150 mM) in water, pH 8.0|
* PMSF is inactivated in aqueous solutions and should be added to buffers immediately before use. The half-life of an aqueous solution of PMSF is around 30 minutes at pH 8.
All these protease inhibitors are normally prepared as stock solutions and stored in aliquots at –20°C. Stock solutions are usually stable for up to six months. In addition, ready-to-use mixtures of protease inhibitors are available. Complete Protease Inhibitor Cocktail Tablets (Roche Molecular Biochemicals) are available in an EDTA-free formulation and are recommended for use during the purification of 6xHis-tagged proteins under native conditions.