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.