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.